Discovery and Characterization of AMPA Receptor Modulators ...

1521-0103/357/2/394–414$25.00 http://dx.doi.org/10.1124/jpet.115.231712THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 357:394–414, May 2016Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Discovery and Characterization of AMPA Receptor ModulatorsSelective for TARP-g8s

Michael P. Maher, Nyantsz Wu, Suchitra Ravula, Michael K. Ameriks, Brad M. Savall,Changlu Liu, Brian Lord, Ryan M. Wyatt, Jose A. Matta, Christine Dugovic, Sujin Yun,Luc Ver Donck, Thomas Steckler, Alan D. Wickenden, Nicholas I. Carruthers,and Timothy W. LovenbergJanssen Research and Development, LLC, Neuroscience Therapeutic Area, San Diego, California (M.P.M., N.W., S.R., M.K.A., B.M.S.,C.L., B.L., R.M.W., J.A.M., C.D., S.Y., A.D.W., N.I.C., T.W.L.); and Janssen Research and Development, a Division of JanssenPharmaceutica NV, Neuroscience Therapeutic Area, Beerse, Belgium (L.V.D., T.S.)

Received December 23, 2015; accepted March 11, 2016

ABSTRACTMembers of the a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) subtype of ionotropic glutamate recep-tors mediate the majority of fast synaptic transmission withinthe mammalian brain and spinal cord, representing attractivetargets for therapeutic intervention. Here, we describe novelAMPA receptor modulators that require the presence of theaccessory protein CACNG8, also known as transmembraneAMPA receptor regulatory protein g8 (TARP-g8). Using calciumflux, radioligand binding, and electrophysiological assays ofwild-type and mutant forms of TARP-g8, we demonstrate thatthese compounds possess a novel mechanism of actionconsistent with a partial disruption of the interaction betweenthe TARP and the pore-forming subunit of the channel. Oneof the molecules, 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol-2-one (JNJ-55511118), had excellent

pharmacokinetic properties and achieved high receptor occu-pancy following oral administration. This molecule showedstrong, dose-dependent inhibition of neurotransmission withinthe hippocampus, and a strong anticonvulsant effect. At highlevels of receptor occupancy in rodent in vivo models, JNJ-55511118 showed a strong reduction in certain bands on electro-encephalogram, transient hyperlocomotion, no motor impairmenton rotarod, and amild impairment in learning andmemory. JNJ-55511118 is a novel tool for reversible AMPA receptor in-hibition, particularly within the hippocampus, with potentialtherapeutic utility as an anticonvulsant or neuroprotectant. Theexistence of a molecule with this mechanism of action demon-strates the possibility of pharmacological targeting of acces-sory proteins, increasing the potential number of druggabletargets.

IntroductionGlutamate is the primary excitatory neurotransmitter in

mammalian brain. The a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) subtype of glutamate recep-tors are ligand-gated ion channels expressed primarily on

postsynaptic membranes of excitatory synapses in the centralnervous system. AMPA receptors (AMPARs) mediate themajority of fast synaptic transmission within the centralnervous system (CNS). Thus, inhibition or negative modula-tion of AMPARs is an attractive strategy for therapeuticintervention in CNS disorders characterized by excessiveneuronal activity. With the notable exception of pore blockers(which are selective for calcium-permeable AMPA receptors;see Stromgaard and Mellor, 2004), no AMPAR inhibitors have

dx.doi.org/10.1124/jpet.115.231712.s This article has supplemental material available at jpet.aspetjournals.org.

ABBREVIATIONS: ACSF, artificial cerebrospinal fluid; AMPA, a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid; AMPAR, AMPA receptor;ANOVA, analysis of variance; CHO, Chinese hamster ovary; CP-465022, 3-(2-Chlorophenyl)-2-[2-[6-[(diethylamino)methyl]-2-pyridinyl]ethenyl]-6-fluoro-4(3H)-quinazolinone hydrochloride; CNS, central nervous system; CT, carboxyl terminus; DMSO, dimethylsulfoxide; DNMTP, delayednon-match to position; EC50, half-maximal effective concentration; ED50, half-maximal effective dose; EEG, electroencephalogram; EMG,electromyogram; EPSC, excitatory postsynaptic current; EX, extracellular domain; FAM, familiar arm; fEPSP, field excitatory postsynapticpotential; f u, unbound fraction; GluA, AMPA subtype of ionotropic glutamate receptor; GYKI-53655, 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride; HABG, HibernateA supplemented with B27 and Glutamax;HEK-293, human embryonic kidney 293; HPMC, hydroxypropyl methylcellulose; J values, indirect dipole-dipole coupling constants; JNJ-55511118,5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol-2-one; JNJ-56022486, 2-(3-chloro-2-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)phenyl)acetonitrile; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LY-395153, N-[4-[1-(propan-2-ylsulfonylamino)propan-2-yl]phenyl]benzamide; MES, maximal electroshock; MWM, Morris water maze; NEW, novel arm; NIH, National Institutes of Health;NMDA, N-methyl-D-aspartate; NREM, non-rapid eye movement; NSB, nonspecific binding; PAM, positive allosteric modulator; PCR,polymerase chain reaction; Philanthotoxin-74, (S)-N-[7-[(4-Aminobutyl)amino]heptyl]-4-hydroxy-a-[(1-oxobutyl)amino]benzenepropanamidedihydrochloride; p.o., per os; PTZ, pentylenetetrazole; RED, Rapid Equilibrium Dialysis; REM, rapid eye movement; SB, specific binding;TARP, transmembrane AMPA receptor regulatory protein; TB, total binding; TM, transmembrane domain.

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http://jpet.aspetjournals.org/content/suppl/2016/03/17/jpet.115.231712.DC1Supplemental material to this article can be found at:

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been found to have selectivity among the AMPAR subtypes, orto exhibit regional specificity. Since AMPAR activity isubiquitous within the CNS, general antagonism results inundesired effects, such as ataxia, sedation, and/or dizziness.In clinical use, AMPAR antagonists have very narrow thera-peutic dosing windows: the doses needed to obtain anticon-vulsant activity are close to or overlap with doses at whichundesired effects are observed (Rogawski, 2011).Over the past two decades, investigations into the quater-

nary structure of native AMPA receptors have revealed aremarkably large set of interaction partners. Heterologousexpression of individual members of the AMPA subtype ofionotropic glutamate receptor (GluA) is sufficient to formfunctional AMPA receptors. However, full recapitulation ofthe trafficking, localization, gating characteristics, andpharmacology of native AMPA receptors requires coassem-bly with a large and diverse set of accessory proteins(Jackson and Nicoll, 2011; Schwenk et al., 2012; Strauband Tomita, 2012). These auxiliary subunits include cyto-skeletal and anchoring proteins, other signaling proteins,and several intracellular and transmembrane proteins withlargely unknown functions. The wide variety of proteinswhich can participate in AMPA receptor complexes vastlyincreases the ability of a neuron to tune the responsecharacteristics of its synapses. Here, we demonstrate thatthese accessory proteins can be used as novel pharmacologicaltargets.Members of the transmembrane AMPA receptor regula-

tory protein (TARP) family (CACNG2, 3, 4, 5, 7, and 8) areassociated with most, if not all, AMPARs in the brain. Theseproteins were originally discovered and named due to theirhomology to the gamma subunit of voltage-gated calciumchannels (Letts et al., 1998; Burgess et al., 1999; Klugbaueret al., 2000). TARPs were subsequently found to associatewith and to modulate the activity of AMPA receptors(Hashimoto et al., 1999; Tomita et al., 2003). Several TARPshave distinct region-specific expression in the brain, leadingto physiologic differentiation of the AMPA receptor activity.It has been theorized that targeting individual TARPs mayenable selective modulation of specific brain circuits withoutglobally affecting synaptic transmission (Gill and Bredt,2011). The expression pattern of TARP-g8 is particularlyattractive in this respect. Based upon in situ hybridizationstudies, TARP-g8 is the predominant TARP throughout thehippocampus, and is expressed within essentially all neu-rons within the stratum pyramidale and stratum granulo-sum. In addition, it is expressed in a substantial proportionof neurons in the amygdala, olfactory bulb, and olfactorynucleus, and in certain layers within the frontal cortex. Incontrast, TARP-g8 shows very little expression within thehindbrain, midbrain, or thalamus (Tomita et al., 2003; Leinet al., 2007; http://mouse.brain-map.org/experiment/show/72108823).Negative modulation of AMPA receptors with a molecule

selective for TARP-g8 offers the possibility of selectivelyreducing excitatory transmission within brain circuits associ-ated with neuropsychiatric or neurologic disorders. Such anagent could be a useful therapeutic in pathologic conditionscharacterized by hyperactivity within the hippocampus—forexample, temporal lobe epilepsy. This approach should miti-gate the side-effect profile attributed to nonselective AMPARantagonists (Ko et al., 2015).

Here, we describe the in vitro and in vivo characterization of5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol-2-one (JNJ-55511118) and 2-(3-chloro-2-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)phenyl)acetonitrile (JNJ-56022486). Thesecompounds are potent negative modulators of AMPA receptorscontaining TARP-g8. They show exquisite selectivity, with nomeasurable effects upon AMPARs containing other TARPs, orupon TARP-less receptors. Using chimeric proteins comprisingvarious segments of TARP-g8 and -g4 followed by site-directedmutagenesis,we identified the specific amino acids responsible forthis remarkable selectivity. We demonstrate in vivo targetoccupancy using ex vivo autoradiography, and provide a pre-liminary investigation of the in vivo pharmacological effects ofTARP-g8–selective AMPA receptor inhibition.

Materials and Methods3-(2-Chlorophenyl)-2-[2-[6-[(diethylamino)methyl]-2-pyridinyl]ethenyl]-

6-fluoro-4(3H)-quinazolinone hydrochloride (CP-465022; Menniti et al.,2000), 1-(4-Aminophenyl)-3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI-53655;Bleakman et al., 1996), and (S)-N-[7-[(4-Aminobutyl)amino]heptyl]-4-hydroxy-a-[(1-oxobutyl)amino]benzenepropanamide dihydrochlo-ride (Philanthotoxin-74; Kromann et al., 2002) were purchased fromTocris (Bristol, UK). N-[4-[1-(propan-2-ylsulfonylamino)propan-2-yl]phenyl]benzamide (LY-395153; Linden et al., 2001) was pur-chased from Diverchim (Roissy-en-France, France). Perampanel(Hanada et al., 2011) was purchased from Alsachim (Illkirch-Graffenstaden, France). Unless otherwise noted, all data analyses,statistics, and data plots were performed using Origin 2015 orOriginPro 2015 (OriginLab, Northampton, MA). Grubbs’ test wasperformed prior to statistical analysis; if identified, a single extremeoutlier was excluded from further analysis. Unless otherwise noted,averages are expressed as the mean 6 S.E.M. Significance levels infigures are denoted as follows: *P , 0.05, **P , 0.01, and ***P ,0.001. Unless otherwise noted, parameters from linear and nonlinearleast-squares fitting procedures are expressed as the value6 standarderror.

Animal studies described in this article that were performed in theUnited States were in accordance with the Guide for the Care andUseof Laboratory Animals (National Research Council, 2011). Studiesperformed in Europe were in accordance with the European Commu-nities Council Directive 2010/63/EU (EuropeanUnion, 2010) and locallegislation on animal experimentation. Facilities were accredited bythe Association for the Assessment and Accreditation of LaboratoryAnimal Care. Animals were allowed to acclimate for 7 days afterreceipt. They were housed in accordance with institutional standards,received food and water ad libitum, and weremaintained on a 12-hourlight/dark cycle.

Chemical Synthesis

General Synthetic Methods. All reagents were purchased fromSigma-Aldrich (St. Louis, MO), Strem Chemicals (Newburyport, MA),or Combi-Blocks (San Diego, CA) and used without further purifica-tion, except where noted. Solvents were purchased from EMDMillipore (Cincinnati, OH) and dried by passing through activatedalumina columns maintained under argon. All reactions were con-ducted under a nitrogen atmosphere unless otherwise noted. Flashchromatography was performed on Teledyne Isco CombiFlash sys-tems using commercially available RediSep silica gel cartridges(Teledyne Isco, Lincoln, NE). Reverse-phase high-performance liquidchromatography purifications were performed on an Agilent 1100Series system (Agilent Technologies, Santa Clara, CA) with a WatersXBridge C18 OBD 5 mM preparative column (Waters Corporation,Milford, MA) unless otherwise noted. NMR spectra were recorded on a

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BrukerUltraShield-400,BrukerUltraShield-500, orBrukerUltraShield-600 spectrometer (Bruker AG, Fallanden, Switzerland) and werereferenced to trimethylsilane. Chemical shifts were recorded in partsper million relative to trimethylsilane, and indirect dipole-dipolecoupling constants (J values) are reported in Hertz. Combustionanalysis was performed at Intertek Pharmaceutical Services (White-house, NJ). Tritium labeling was conducted at Moravek Biochemicals(Brea, CA). The reaction scheme for the synthesis of JNJ-55511118 isshown in Supplemental Fig. 1. The reaction scheme for the synthesisand tritiation of JNJ-56022486 is shown in Supplemental Fig. 2.

Molecular Biology

Molecular Cloning of GluA Receptors and Their AccessoryProteins from Different Species. cDNAs for human GluA1-FLIP;GluA1-FLOP; GluA2-FLOP; GluA2-FLIP; GluA3-FLOP; GluA4-FLOP; and and monkey, dog, mouse, and rat GluA1-FLOP, as wellas their accessory proteins, including human CACNG2, CACNG3,CACNG4, CACNG7, CACNG8, CNIH2, monkey CACNG8, mouseCACNG8, and CACNG8, were polymerase chain reaction (PCR)amplified from brain cDNAs from respective species. A pointmutationwas introduced into the GluA2 constructs at the Q/R editing site toallow calcium permeability in the expressed protein (Burnashev et al.,1992). Dog CACNG8 was synthesized with codon optimization basedon the published sequence (GenBank accession no. KT749896). Thesequences for PCR primers are listed in Supplemental Table 1. ThePCR products were cloned into mammalian expression vectors asindicated: pCIneo (Promega, Madison, WI), pcDNA3.1(1) (Life Tech-nologies, Carlsbad, CA), or pcDNA4/TO (Life Technologies). Cloningsites (highlighted in shaded letters) were introduced into primers tofacilitate the cloning process. The insert regions were sequenced toconfirm the sequence identities. FLOP and FLIP splice variants aredesignated with o and i suffixes, respectively (e.g., the FLIP variant ofGluA1 is designated GluA1i).

Generation of GluA1o-CACNG8 Fusion Protein ExpressionConstructs. To ensure a 1:1 stoichiometry of GluA1o and g8 in theexpressed channel, a fusion of the cDNAs for GRIA1o and CACNG8was used. Following Shi et al. (2009), we fused the cDNA encodingthe C terminus of GluA1o to the cDNA encoding theN terminus of g8.We inserted a linker sequence encoding QQQQQQQQQQEFATbetween the two full-length cDNAs. The channels expressed withthis construct appear to have identical properties to channels formedby coexpression of GRIA1o with an excess of CACNG8 (Shi et al.,2009). Human, mouse, and rat GluA1o-CACNG8 fusion proteinexpression constructs were generated by overlapping PCR followedby cloning into mammalian expression vectors. The human GluA1o-CACNG8 fusion protein expression DNA was cloned into pCIneobetween EcoR1 and Not1 sites, whereas the mouse and rat GluA1o-CACNG8 expression constructs were cloned into pcDNA4/TO be-tween HindIII and Not1 sites. The primers and templates for theoverlapping PCRs are listed in Supplemental Table 2. All cloneswere sequenced, and the identities were confirmed. DNA coding andpredicted amino acid sequences for the fusion constructs are listed inSupplemental Table 3.

Construction of Chimeric Proteins Using TARPs g8, g4, andg2. The sequences for the human variants of each protein werealigned using the UniProt alignment tool, which also predicted thetransmembrane segments of the proteins. The protein sequences weredivided into nine regions separated near the borders of the predictedtransmembrane sections; these nine regions were the N and C termini(CT), the four transmembrane domains (TM1–TM4), the two extra-cellular domains (EX1, EX2), and the intracellular domain. Thepredicted topology of the TARP is shown in Fig. 3A, and the splicepoints between the TARPs are shown in Supplemental Table 4. Thechimeras were designated by a nine-digit number; each digit indicatesthe TARP used for that section of the protein, starting from theN terminus. Graphical representations of the chimeric TARPs areshown in Supplemental Fig. 3. The chimeric expression constructs

were generated using overlapping PCRs, except those indicatedotherwise. First, two separate PCR reactions (59 end PCR and 39 endPCR) that generated overlapping PCRproductswere performed.Next,the 59 end and 39 end PCR products were mixed to serve as thetemplate for the PCR reactions that generated the full-length PCRproduct for molecular clonings. The primers and templates used forPCR reactions are listed in Supplemental Table 5. DNA coding andpredicted amino acid sequences for the chimeric constructs are listedin Supplemental Table 6.

Generation of Point Mutations. All mutant expression con-structs were generated by overlapping PCR using the human wild-type CACNG8 or CACNG4 cDNA as the template. The primers usedfor generation of themutants are listed in Supplemental Table 7. DNAcoding and predicted amino acid sequences for the chimeric constructsare listed in Supplemental Table 8.

Calcium Flux Assay

A clonal cell line stably expressing the human GluA1o-g8 fusionconstruct under geneticin selection in human embryonic kidney 293(HEK-293) cells was established for the primary calcium flux assay.All other combinations of GluA subunits and TARPs were performedusing cotransfections of the respective plasmids into HEK-293-F cells.AMPA receptors formed by cotransfections are designated with theplus symbol (e.g., GluA1i cotransfected with TARP-g8 is referred to asGluA1i1g8).

For assays with transiently transfected cells, the cells weregenerated by bulk transfection. Prior to transfection, 293-F cells werecultured in FreeStyle-293 Expression Medium (Gibco, Grand Island,NY) at 0.5–2 million cells/ml in shaker flasks at 37°C and 8% CO2 at120 rpm. At the time of transfection, cells were diluted to 1 million/mlwith FreeStyle-293 medium. Cell viability was above 90% for trans-fections to be considered successful. Transfection was performed bycombining equal amounts of pAdvantage vector (Promega) and targetDNA. Total DNA was 50 mg per 40-ml transfection. The DNA ratio ofAMPA receptor to TARPs was 4:1. The transfection reagent wasFreeStyle MAX (Invitrogen, Carlsbad, CA). Cells were seeded into384-well polylysine-coated plates at 15,000 cells/well at 16–24 hoursafter transfection, and used for assays 24–48 hours after transfection.

The calcium flux assays were performed as follows. Cell plates werewashed with assay buffer (135 mM NaCl, 4 mM KCl, 3 mM CaCl2,1 mM MgCl2, 5 mM glucose, and 10 mM HEPES, pH 7.4, 300 mOsm)using a Biotek EL405 plate washer (Biotek, Winooski, VT). The cellswere then loaded with a calcium-sensitive dye according to themanufacturers’ instructions (Calcium-5 or Calcium-6; Molecular De-vices, Sunnyvale, CA) combined with the test compounds at a range ofconcentrations. Calcium flux following the addition of 15 mM gluta-mate was monitored using a FLIPR Tetra (Molecular Devices).

The fluorescent response in each well was normalized to theresponse of negative and positive control wells. The negative controlwells had no added compounds, and the positive control wells had beenincubated with 50 mM CP-465022 (a non–subtype-selective AMPARantagonist; Lazzaro et al., 2002). The responses (R) to glutamate asfunctions of the test compound concentrations (x) were fitted to a four-parameter logistic function (eq.1):

R5A2 1 ðA1 2A2Þ=�11 ðx=x0Þp

�(1)

The fitted parameter corresponding to the midpoint (ϰ0) was takento be the potency of inhibition of the compound (IC50; 50% inhibitoryconcentration). Potency is expressed as Equation 2:

pIC50 5 2 log10ðIC50½M�Þ; ð2Þwhere pIC50 is the negative log of the 50% inhibitory concentration.

Knockout Animals

The TARP-g8 knockout mouse line Cacng8tm1Ran was originallydescribed by Rouach et al. (2005). This mouse line, generated by

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homologous recombination in embryonic stem cells to replace exons 2and 3with a neomycin resistance gene, was rederived by back-crossinginto a C57BL/6J mouse line at Jackson Laboratory (Bar Harbor, ME).

Patch-Clamp Electrophysiology

Heterologous Cells. Studies with GluA1i were performed withtransiently transfected HEK-293 cells. Human GluA1i with orwithout human TARP-g8 was transfected into HEK-293 cells usingLipofectamine 2000 (Life Technologies) following the manufac-turer’s instructions. Eight to 24 hours after transfection, cells wereplated onto 12-mm glass coverslips in Dulbecco’s modified Eagle’smedium with high glucose, without glutamine (Sigma-Aldrich),supplemented with 10% fetal bovine serum, and incubated at 37°Cin a humidified 5% CO2 incubator. To increase surface expression,cells were transferred to a humidified 5% CO2 incubator at 30°C for6–24 hours immediately prior to use. Recordings were performed48–72 hours post-transfection.

For patch-clamp electrophysiology on heterologously expressedGluA1o-g8 and human GluA1o-g2, we established single-cell clonesstably expressing these constructs inChinese hamster ovary cells (T-Rex-CHO; Invitrogen) using a tetracycline-inducible expression vector.Cells were cultured in Ham’s F-12 supplemented with 10% fetalbovine serum, 100 mg/ml zeocin, and 5 mg/ml blasticidin. To induceexpression, 1 mg/ml tetracycline was added to the culture medium 1–4days prior to use. Cells were plated onto 12-mm plain glass coverslipsand incubated at 37°C in a humidified 5% CO2 incubator. To increasesurface expression, cells were transferred to a humidified 5% CO2

incubator at 30°C for 6–24 hours immediately prior to use.Hippocampal Neurons. Acute hippocampal neurons were

obtained from 8- to 12-week old C57BL/6J male mice, following theprotocol described by Brewer (1997) with the following modifications.Medium was prepared by supplementing HibernateA with 2% B27and 0.5 mM Glutamax (HABG medium; all reagents from LifeTechnologies). Mice were asphyxiated with CO2 and then decapitatedin accordancewithNational Institutes ofHealth (NIH) animal and useguidelines. The brain was rapidly removed, then placed into ice-coldHABG medium. Sagittal slices, 300 mm thick, were obtained using aVT1200S microtome (Leica Biosystems, Buffalo Grove, IL). Sliceswere cut in ice-cold solution composed of 150 mM sucrose, 50 mMNaCl, 25 mM NaHCO3, 10 mM glucose, 7 mM MgSO4, 2.5 mM KCl,1.25 mM Na3PO4, and 0.5 mM CaCl2 equilibrated with 95% O2 and5% CO2. The hippocampus was isolated from the rest of the slices andtransferred to a calcium-free HibernateA Minus Calcium solution(BrainBits, Springfield, IL) containing 20 mg of papain (WorthingtonBiochemical, Lakewood, NJ) and 0.5 mM Glutamax (Life Technolo-gies) and digested at 30°C under gentle shaking for 30 minutes. Then,the papain solution was aspirated and replaced with HABG. Sliceswere gently triturated with fire-polished Pasteur pipettes. The su-pernatant containing dissociated neurons was collected, and thencentrifuged for 2 minutes at 200g. The cell pellet was collected andthen resuspended in HABG. The cell suspension was then plated overcoverslips, and isolated neurons were picked under visual inspectionfor whole-cell patch-clamp recordings. The extracellular and intracel-lular solutions were the same as described earlier for transfectedHEK-293 cells.

Cerebellar Granule Cells. Cerebellar granule cell cultures wereprepared following Brewer (1997) with the following modifications.Cerebella were harvested from newborn Sprague-Dawley rat pups(1–4 days old, males and females were mixed). Tissue was mincedmanually prior to trypsin digestion. Dissociated cells were plated ontoglass coverslips coated with poly-D-lysine and fibronectin, and thencultured for 2–4 weeks prior to use.

Electrophysiology. Whole-cell and outside-out patch electrophys-iology (Hamill et al., 1981) was performed using 1.5-mm-diameter glasscapillary tubes (TW150-4; World Precision Instruments, Sarasota, FL)pulled to a fine tip with a Sutter P-97 micropipette puller (SutterInstruments, Novato, CA). The intracellular buffer was 90 mM

potassium fluoride, 30 mM KCl, 10 mM HEPES, and 5 mM EGTA (pH7.4, 290 mOsm). The extracellular buffer was 135 mM NaCl, 4 mMKCl, 2mMCaCl2, 1mMMgCl2, 5mMglucose, and 10mMHEPES (pH7.4, 300 mOsm). The open-tip resistances of the micropipettes usingthese solutions were 2–4 MV. Recordings were performed in voltage-clamp mode using an Axopatch 200B amplifier and Digidata 1440Adigitizer (Axon Instruments, Sunnyvale, CA). Recordings were con-trolled and measured using pClamp 9.2 software (Axon Instruments).Current was measured by holding the interior of the cell at 260mV,using a 5-kHz low-pass filter. The cells were continuously perfusedthrough 7-mm square glass barrels using a solenoid-controlled solu-tion switching device (PF-77B; Warner Instruments, Hamden, CT).The peak current in response to a 500-ms exposure to 10 mMglutamate every 5 seconds was measured before and after exposureto test compound; 10 mM glutamate was chosen as a saturatingconcentration for the peak responses (Robert and Howe, 2003).Steady-state currents were measured during the last 50 ms of theglutamate application. Upon establishing stable glutamate-evokedresponses, JNJ-55511118 was applied before and during glutamateapplication until a steady-state inhibition was observed (typically50–60 seconds). For analysis, the mean peak current of five traces inthe presence of test compound was divided by the mean peak currentof five traces prior to the addition of test compound.

For ultra-fast glutamate perfusion, a piezo-driven perfusion systemwas used (Siskiyou, Grants Pass, OR). Recordings on outside-outpatches were performed using an AxoPatch 200B amplifier (AxonInstruments), and signals were filtered at 10 kHz and digitized at50 kHz. Data acquisition and online analysis were performed usingpClamp 9 (Axon Instruments). Current decay kinetics were fitted witha double exponential function using Origin (OriginLab, Northampton,MA) and expressed as a weighted decay time constant. For recoveryfrom desensitization, an initial desensitizing pulse of glutamate wasfollowed by a second pulse of glutamate at varying time intervals. Therecovery from desensitization was expressed as the current peakamplitude fraction of the second pulse to the first pulse at a given timeinterval, and was fitted using a single exponential function (for TARP-g8–containing AMPA receptors) or a double exponential function (forTARP-less AMPA receptors) using Origin (OriginLab).

Brain Slice Whole-Cell Patch Clamp Electrophysiology

Male mice (2–3 weeks) were anesthetized with isoflurane and thendecapitated in accordance with NIH animal care and use guidelines.Transverse hippocampal slices (300mmthick) were cut in ice-cold highsucrose buffer containing 87 mM NaCl, 2.5 mM KCl, 0.5 mM CaCl2,7 mM MgSO4, 1.25 mM NaH2PO4, 25 mM NaHCO3, 25 mM glucose,and 75mM sucrose equilibrated with 95%O2 and 5% CO2. Slices werethen placed in artificial cerebrospinal fluid (ACSF) at 35°C for30 minutes, and then allowed to recover for at least 1 hour in ACSFat room temperature. ACSF for electrophysiological recordings con-tained 119 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgSO4,1 mM NaH2PO4, 26.2 mM NaHCO3, and 11 mM glucose equilibratedwith 95% O2 and 5% CO2. The intracellular recording solutioncontained 130 mM CsMeSO4, 10 mM HEPES, 8 mM KCl, 4 mMMgATP, 0.4 mM NaGTP, 10 mM sodium creatine, and 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid.

Excitatory postsynaptic currents (EPSCs) intracellularly recordedfrom a neuron in the CA1 pyramidal cell layer were evoked byelectrical stimulation of the Schaffer collateral/commissural pathwayusing a monopolar glass stimulating electrode (filled with ACSF)placed in the stratum radiatumof CA1 (0.1-Hz stimulation frequency).In caseswhere trains of five stimulations at 50Hzwere done, the pulsetrain was alternated with one stimulation (0.1 or 0.05 Hz betweentrains). Test compounds were bath-applied. To evoke AMPA EPSCs,neurons were held at270 mV.N-methyl-D-aspartate (NMDA) EPSCswere recorded at 140 mV 50 ms after the AMPA EPSC at 270 mV.NMDA EPSCs were recorded prior to and after establishing thesteady-state inhibition of peak AMPA EPSCs. Recordings were


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performed using a Multiclamp 700B patch-clamp amplifier (AxonInstruments); signals were filtered at 4 kHz, digitized at 10 kHz, anddisplayed and analyzed online using pClamp 9.2 (Axon Instruments).

In some experiments, trains of 50-Hz stimulation were used todetermine the paired-pulse ratio. In this case, theEPSCmagnitude foreach pulse was measured from the baseline current immediately afterthe pulse to the peak current. The paired-pulse ratio was calculated asthis EPSC magnitude, divided by the immediately preceding EPSCmagnitude.

Brain Slice Field Excitatory Postsynaptic PotentialsElectrophysiology

Male mice (7–9 weeks) were anesthetized with isoflurane and thendecapitated in accordance with NIH animal care and use guidelines.Horizontal hippocampal slices (300mmthick)were cut in ice-cold high-sucrose ACSF (composition described earlier). Slices were then placedin ACSF at 35°C for 30 minutes, and then allowed to recover for atleast 1 hour in ACSF at room temperature. Slices were then placedon a perforated multielectrode array chip (Multichannel Systems,Reutlingen, Germany) and perfused with ACSF heated to 35°C.Excitatory postsynaptic potentials were evoked with electrodesplaced in the CA1 radiatum once every minute, sampling at 50 kHz.Multielectrode array responses across multiple electrodes were chosenbased on stability of the response and amplitude (.0.4 mV), which werethen averaged to generate anN5 1 for each slice. Test compounds werebath-applied.

Radioligand Binding

Male Sprague-Dawley rats (6–14 weeks) were anesthetized withisoflurane and then decapitated in accordance with NIH animal careand use guidelines. The brain was removed, and hippocampi wererapidly dissected and then frozen at 280°C until use. For each assay,two hippocampi per five 96-well plates were used. On the day of theexperiment, the hippocampal tissue was thawed, and then homoge-nized in assay buffer (50mMTris, pH 7.4) for 30 seconds at high speed.The homogenate was centrifuged at 1500 rpm for 5 minutes followedby careful decanting of the supernatant, which was centrifuged at39,000g for 30 minutes. Ice-cold assay buffer was added to the cellpellet. The protein concentration within the pellet was determined bycolorimetry using a Pierce bicinchoninic acid protein assay kit(Thermo Fisher Scientific, Rockford, IL), then diluted with assaybuffer to obtain a concentration of 200–400 mg protein per milliliter(10–20 mg protein per well).

Binding assays were performed in Whatman GF/B 96-well filterplates (GE Healthcare, Little Chalfont, United Kingdom) presoakedwith 0.3% polyethylenimine. When manufactured, the stock solutionof tracer was 34.5 mM single-labeled [3H]JNJ-56022486 and 10.3 mMunlabeled JNJ-56022486 in ethanol. The actual stock concentrationwas calculated at the time of use based upon the decay rate of tritium.Tenmicroliters of 10� test compound, 40ml of 2.5� tracer, and 50ml ofmembrane homogenate were placed into each well. The reaction wasincubated for 2 hours at 4°C on a shaker, then terminated by filtrationfollowed by washing with ice-cold assay buffer four times. After dryingfor 30 minutes at 50°C, 60 ml/well MicroScint-O (PerkinElmer,Waltham, MA) was added to the plate. Radioactivity retained on thefilters was measured using a TopCount liquid scintillation counter(PerkinElmer, Waltham, MA). The scintillation counter was cali-brated with a linear least-squares fit to the radioactivity counts fromknown quantities of [3H]JNJ-56022486. All analyses were performedin Origin 2015 (OriginLab).

For saturation binding experiments, a 2� serial dilution of [3H]JNJ-56022486 in quadruplicate wells was used for total binding (TB),with 50 mMJNJ-55511118 in quadruplicate wells for determination ofnonspecific binding (NSB). Ligand depletion was determined bycomparing the radioactivity counts of total binding to the counts in aseparate plate spiked with an equivalent amount of radioligand;

ligand depletion was ,15% at each concentration. Specific binding(SB) at each radioligand concentration was calculated as SB 5TB – NSB at each radioligand concentration. NSB was fitted to alinear function using linear least-squares analysis. SB was con-verted to pmol/mg protein, and then fitted to a single-site bindingmodel to determine the dissociation coefficient (KD) and totalreceptor concentration (Bmax):

SB5Bmax½hot�

½hot�1 ½cold�1KD

For competition binding experiments, a serial dilution of the testcompound was prepared in assay buffer and combined with a finalconcentration of 20 nM radioligand. NSB was determined using eightwells containing a blocking concentration of 50 mM JNJ-55511118along with radioligand and tissue membranes, and total binding wasdetermined using eight wells containing only radioligand and tissuemembranes. Four replicates for each test compound concentrationwere used. After incubation and washing as described earlier, theradioactive counts (SB) in each well were normalized to the total andnonspecific binding counts and then fitted to a single-site logisticfunction. The equilibrium dissociation constant (Ki) was calculatedfrom the midpoint parameter of the fit, adjusted for radioligandconcentration using the Cheng-Prusoff correction (Cheng and Prusoff,1973).

Plasma Protein Binding

Plasma protein binding was determined by equilibrium dialysisusing the Rapid Equilibrium Dialysis (RED) device (Thermo FisherScientific), consisting of a Teflon base plate, and RED Device insertscomprising two (sample and buffer) side-by-side chambers separatedby a dialysis membrane (molecular weight cut-off � 8000). Compoundswere prepared as 100 mM dimethylsulfoxide (DMSO) stocks andspiked into 1 ml of mouse, rat, and human plasma (BioreclamationIVT,Westbury, NY) to make a final concentration of 1 mM. Plasma (300 ml)was dispensed into the sample well, and dialysis buffer (100 mMpotassium phosphate, pH 7.4, 500 ml) was dispensed into the bufferwell. Each compound was tested in triplicate. The RED device wassealed, and equilibrium was permitted for 6 hours in a 37°C incubatorwith gentle agitation at 100 rpm. After incubation, plasma samples wereprepared by transferring 10ml fromplasmawells to 90ml of freshdialysisbuffer, and buffer samples were prepared by transferring 90 ml frombuffer wells to 10 ml of naïve plasma. In addition, a reference samplewithout equilibration was prepared in triplicate by mixing 10 ml ofplasma containing 1 mM compound with 90 ml of buffer to determinecompound recovery from the assay. Two volumes of 1:1 acetonitrile:methanol spiked with the internal standard phenytoin (0.2 mg/ml) wasadded to the reference and samples. Precipitation of plasma proteinbinding was allowed for 15 minutes before the reference and sampleswere centrifuge clarified. Supernatant (10 ml) was used for liquidchromatography–tandem mass spectrometry (LC-MS/MS) analyses.

Brain Tissue Binding

Brain tissue binding was assessed by an equilibrium dialysistechnique similar to the procedure described for plasma proteinbinding. Rat brain tissue homogenate prepared in phosphate-buffered saline buffer [pH 7.4, 1:10 (w/v)] was spiked with compoundDMSO stock solution to yield a final concentration of 5 mM. Thedialysis was carried out in a shaking incubator at 37°C for 5 hours.After incubation, 25 ml of homogenate or 50ml of buffer was extractedwith 50 ml of DMSO and 300 ml of acetonitrile and analyzed byLC-MS/MS using the calibration curves across an appropriateconcentration range and quality control samples. All determinationswere conducted in triplicate. The apparent unbound fraction (f u,app)was determined as the ratio of the concentration measured in thehomogenate to the concentration measured in the buffer. Theunbound fraction in undiluted brain was calculated as

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fu;brain 5fu;app

D1 fu;app 2Dfu;app;

where D is a dilution factor of 10. Subsequently, the percentage ofcompound bound to brain tissue (%BTB) was determined as

%BTB5�12 fu;brain

�� 100%:

Pharmacokinetic Studies

Single-dose pharmacokinetic studies of JNJ-55511118 in maleSprague-Dawley rats were conducted by BioDuro, LLC (Beijing,China) following i.v. (1 mg/kg) and per os (p.o.; 5 mg/kg) administra-tion as a solution in 20% hydroxypropyl-b-cyclodextrin with threeequivalents of sodiumhydroxide. Bloodwas sampled at predose and at0.033 (i.v.), 0.083 (i.v.), 0.25, 0.5, 1, 2, 4, 8, and 24 hours postdose.Plasma concentrations were quantitated by LC-MS/MS. Pharmacoki-netic parameters were derived fromnoncompartmental analysis of theplasma concentration versus time data using WinNonlin software(Pharsight, Palo Alto, CA).

Single-dose pharmacokinetic studies of JNJ-55511118 in male C57/BL6mice were conducted by BioDuro, LLC following p.o. (10 mg/kg) admin-istration as a suspension in 0.5% hydroxypropyl methylcellulose(HPMC). Blood was sampled at predose and at 0.5, 1, 2, 4, 8, and24 hours postdose.

Blood-Brain Barrier. Adult male animals were dosed by oraladministration of a suspension in HPMC. The animals were eutha-nized using carbon dioxide and decapitated at specified time pointsafter drug administration. Brains were rapidly frozen on powdereddry ice and stored at 280°C before sectioning for receptor occupancystudies or for compound concentration determination by LC-MS/MS.The blood-brain barrier ratio was calculated as the compoundconcentration in the brain divided by the concentration in the plasmafor each animal.

LC-MS/MS. JNJ-55511118 was quantified on an API4000 MS/MSSystem (Applied Biosystems, Concord, Ontario, Canada) interfacedwith an Agilent 1100 Series high-performance liquid chromatogra-pher. Samples were loaded onto a 2.1 � 30-mm ACE 5mm C4 100Acolumn (Advanced Chromatography Technologies Ltd., Aberdeen,Scotland) under a flow rate of 0.9 ml/min using 5 mM ammoniumacetate (0.1% formic acid) as mobile phase A and acetonitrile (0.1%formic acid) as mobile phase B. Starting with 87% mobile phase A for0.4 minute, mobile phase B was increased from 13 to 90% using a lineargradient for 0.8 minute, held at 90% B for 0.3 minute, and equilibratedat 13% B for 1.0 minute for an overall run time of 2.5 minutes. JNJ-55511118 was quantified by MS/MS in the positive ion mode bymonitoring the transition of 328.95 to 208.10 m/z.

Ex Vivo Receptor Occupancy

Receptor occupancy was assessed by ex vivo autoradiography usingthe TARP-g8 receptor antagonist [3H]JNJ-56022486. Coronal andsagittal tissue sections of 20-mm thickness were prepared for autora-diography as previously described (Langlois et al., 2001). Tissuesections were incubated for 10 minutes in 50 mM Tris HCl containing0.1% bovine serum albumin (pH 7.4) with 5nM [3H]JNJ-56022486 atroom temperature. Nonspecific binding was characterized with astructurally distinct TARP-g8 receptor antagonist. Sections wererinsed in 50 mM Tris HCl containing 0.1% bovine serum albumin onice four times for 10 minutes per rinse, followed by two dips in ice-colddeionized water, then dried under a stream of cold air. Digitizedimages were acquired with b-Imager DFine or TRacer (Biospacelab,Paris, France).

In Vivo Electrophysiology

Male Sprague-Dawley rats (Charles River Laboratories, San Diego,CA) weighing approximately 300–450 g were used for these experi-ments. The jugular vein was precannulated by the vendor to facilitate

intravenous administration of compound. Animals were singly housed,given food and water ad libitum, andmaintained on a 12-hour light anddark cycle.

Evoked population spikes from the CA1 region of the hippocampuswere recorded following established procedures (Jeggo et al., 2014)with the following modifications. Animals were anesthetized withisoflurane for the duration of the surgical preparation and recordingperiods while body temperature wasmaintainedwith a homeothermicheating pad. A small piece of skull overlaying the hippocampus wasremoved using a hand-held drill before a concentric bipolar stimulat-ing electrode (FHC, Bowdoin, ME) and tungsten recording microelec-trode (World Precision Instruments) were inserted into CA1 using thefollowing stereotaxic coordinates (from the bregma):

Stimulating electrode: anterior-posterior 5 3.4 mm, medial-lateral 5 2.75 mm

Recording electrode: anterior-posterior 5 4.4 mm, medial-lateral 5 2.25 mm.

Electrodes were typically inserted to a depth of 2–2.5 mm below thepial surface before test stimuli were used to help optimize the evokedsignal and determine the final recording depth. Stimulation intensi-ties evoking a 30–60% maximal response were used. Signals from therecording electrode were amplified and filtered (1 Hz to 10 kHz,DAM80 bio-amplifier; World Precision Instruments), then digitizedand collected (40-kHz sampling) using a PowerLab 16/35 dataacquisition unit controlled by LabChart Pro software (ADInstru-ments, Colorado Springs, CO). Brief stimulus pulses were continu-ously delivered to the hippocampus at a rate of 0.33Hz, and the evokedneural responseswere recorded. A stable baseline period of 10minuteswas obtained before administration of the compound, and evokedresponses were obtained for an additional 60minutes thereafter. JNJ-55511118 was formulated in 5% N-methyl-2-pyrrolidone plus 20%Cremophor (BASF, Ludwigshafen, Germany) plus 75% water anddosed intravenously. At the end of the recording session, the brain waselectrically lesioned to determine the final positions of the stimulatingand recording electrodes. Additionally, the brain was removed andplasma samples collected to determine in vivo concentrations ofcompound via LC-MS/MS. The population spike amplitude for eachstimulus was extracted from the recording following the procedureoutlined by Jeggo et al. (2014), normalized to the mean baselineamplitude, then averaged for each dose group according to timerelative to the injection of drug.

Electroencephalogram Recording and Locomotor ActivityStudies in Rats

Experiments were conducted in male Sprague-Dawley rats(350–450 g; Harlan Laboratories, Livermore, CA). Animals werechronically implanted with telemetric devices (PhysioTel F40-ETT;Data Sciences International, St. Paul, MN) for the recording ofelectroencephalogram (EEG) with two epidural electrodes placed inthe frontal and parietal cortex, electromyogram (EMG), and locomotoractivity as described previously (Dugovic et al., 2009). EEG and EMGsignalswere digitized at a sampling rate of 100Hz.High- and low-passfilters were set at 1 and 30 Hz for the EEG signal. Polysomnographicwave forms were analyzed per 10-second epoch and classified as wake,non-rapid eye movement (NREM), or rapid eye movement (REM)sleep using the computer software program SleepSign (Kissei Comtec,Nagano, Japan). EEG activity within specific vigilance states wasdetermined by power spectral analysis (fast Fourier transform) withina frequency range of 1–30 Hz. Values for power spectra were dividedinto four frequency bands: delta (1–4 Hz), theta (4–10 Hz), alpha/sigma(10–15 Hz), and beta (15–30 Hz). Locomotor activity counts wereanalyzed into 1-minute bins and averaged into 5-minute intervals foreach animal. All results were averaged and expressed as the mean 6S.E.M. in defined time intervals for each animal. To determinewhether differences were significant at a given interval, either aone-way analysis of variance (ANOVA) or two-way repeated-measures


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ANOVA followed by Dunnett’s multiple comparison test wasperformed.

Anticonvulsant Studies

Anticonvulsant studies were performed by NeuroAdjuvants, Inc.(Salt Lake City, UT). Unless otherwise noted, male albino CF1 mice(Charles River Laboratories, Portage, MI) were used as experimentalanimals. All animals were allowed free access to both food and waterexcept when they were removed from their cages for the experimentalprocedure. All mice were housed, fed, and handled in a mannerconsistent with the recommendations of the National ResearchCouncil (2011). No insecticides capable of altering hepatic drug-metabolizing enzymes were used in the animal facilities. All animalswere euthanized in accordance with the Institute of LaboratoryResources policies on the humane care of laboratory animals. All testsubstances were administered orally in 0.5% methylcellulose in avolume of 10 ml/kg body weight. For the maximal electroshock(MES), 6 Hz, and corneal kindling assays, a drop of anesthetic/electrolyte solution (0.5% tetracaine hydrochloride in 0.9% saline)was applied to the eyes of each animal prior to placement of thecorneal electrodes.

Mouse 6 Hz Psychomotor Seizure and MES Tests. Details ofthe procedures have been described previously (Barton et al., 2001;Rowley and White, 2010). In brief, an acute seizure was induced viaelectrical stimulation through electrodes applied to the corneas of testanimals. For the 6 Hz tests, the stimulus was 32 or 44mA at 6 Hz for 3seconds, and the induced seizure was characterized by jaw chomping,vibrissae twitching, forelimb clonus, and Straub tail. For the MEStest, the stimulus was 50 mA at 50 Hz for 0.2 second, and the inducedseizures were characterized by a tonic hindlimb extension. Cohorts ofeight mice for each test concentration and stimulation intensity weretreated with a single oral dose 2 hours prior to challenge with theelectrical stimulation. Mice not displaying the described seizurephenotypes were considered protected.

Mouse Corneal Kindling. The corneal kindling assay followedestablished procedures (Matagne and Klitgaard, 1998; Rowley andWhite, 2010). Kindling was achieved by twice-daily corneal stimu-lation (3 mA, 3 seconds, 60 Hz) until all mice reached an establishedcriteria of five consecutive secondarily generalized seizures (Racinestage 5). For compound testing, a cohort of eight fully kindled micewere administered a single oral dose of the test compound 2 hoursprior to challengewith the kindling stimulus. TheRacine seizure score(0–5; Racine, 1972) was recorded for each mouse and averaged.Animals with seizure scores of 3 or lower were considered protected.

Timed Intravenous Infusion of Metrazol Test. A single dose ofeach test compound or vehicle was administered p.o. to cohorts of10 mice 2 hours prior to the test. Mice were challenged with 0.5%heparinized Metrazol solution [5 mg/ml; pentylenetetrazol (PTZ), Sigma-Aldrich, St. Louis, MO], infused at a constant rate of 0.34 ml/min into alateral tail vein of an unrestrained mouse (Orloff et al., 1949; Whiteet al., 1997). The time in seconds from the start of the infusion to theappearance of the “first twitch,” and then to the onset of sustainedclonus, was recorded. The times to each endpoint were converted tomg/kg of PTZ for each mouse, taking into account the rate of infusion,concentration of PTZ, and weight of the animal.

Amygdala Kindling. Male Sprague-Dawley rats (Charles RiverLaboratories) were surgically implanted with stimulation/recordingelectrodes unilaterally into the amygdala according to the proceduredescribed by McNamara (1995). Rats received daily subthresholdstimulation followed by behavioral and electrographic monitoring fora period of 2–3 weeks, during which time a majority of rats wereconsidered fully kindled (five generalized seizures, Racine scale 4–5,over a period of 8 days). Fully kindled rats received either vehicle(0.5% HPMC) or JNJ-55511118 (suspension in 0.5% HPMC, oralgavage, 10 ml/kg). They were then challenged 2 hours later with thesame kindling stimulation, and their behavioral seizure score andelectrographic after-discharge duration were recorded.

Rotarod

Immediately prior to seizure testing, all mice were subjected to arotarod test to assess motor coordination. Animals were placed on a1-inch knurled rod that rotates at a speed of 6 rpm. The animal wasconsidered motor-impaired if it fell off this rotating rod three timesduring a 1-minute period.

Morris Water Maze

The procedure followed the water maze task described by Atchaet al. (2009), with the following modifications. Video tracking software(EthoVision XT 9.0; Noldus, Wageningen, The Netherlands) was usedto measure the path, time taken, and swim speed for each animal toreach the platform. Male Long-Evans rats (Janvier, Le Genest-Saint-Isle, France; N 5 12 per dose group) were trained for 4 days in threedaily trials with random starting positions to find the hidden platform(days 1–4). The location of the hidden platform was maintainedthroughout the study. When an animal failed to find the platformwithin 60 seconds, it was guided to the platform and allowed to staythere for another 5–10 seconds. Directly after the last acquisition trialon day 4, the animals were subjected to a probe trial for 60 seconds,during which the platform was removed.

Statistical analyses were performed for the averages per day andper trial. For analysis of “latency to platform,” a Cox proportionalhazards model was used. For the other parameters, a repeated-measures ANOVA model was used. For probe trials and percent-per-quadrant measures in acquisition, one-way ANOVA statistics wererun using InVivoStat software (Clark et al., 2012; http://invivostat.co.uk/), with dose used as the treatment factor.

V-maze

Male Long-Evans rats (body weight 250–300 g; Janvier Laborato-ries) were individually housed for 7 days before testing and habituatedto the experimental procedures. The apparatus consisted of twoenclosed arms with walls of different visual contexts positioned at a90° angle to each other, and connected to a center zone (Embrechts andVer Donck, 2014). A top-mounted video camera recorded the move-ments of the animals, and images were analyzed for distance madeand time spent in each arm using EthoVision XT 9.0 (Noldus).

Two hours after dosingwith vehicle or test compound, animals weresubjected to a habituation trial (T1), during which one arm was closedand the other armwas free to be explored for 5minutes [defined as thefamiliar arm (FAM)]. Then during the retention trial (T2), the novelarm was opened (NEW arm), and the animal explored both arms ofthe maze for 5 minutes. The discrimination index indicating prefer-ence for the novel arm was calculated from the time spent in eacharm of the maze during the retention trial: discrimination index 5(NEW 2 FAM)/(NEW 1 FAM).

Statistical analyses were performed using “InVivoStat” software(Clark et al., 2012; http://invivostat.co.uk/). The discrimination indexwas analyzed using single measures parametric analysis, and theoverall effects were determined using ANOVA followed by all-to-onecomparisons without adjustment for multiplicity (Fisher’s least-significant differences tests).

Delayed Nonmatch to Position

Standard operant chambers (Med Associates, St. Albans, VT) wereused. One wall was equipped with two retractable response levers andstimulus lights. The opposite wall contained the reward magazine,equipped with a reward light and an infrared sensor. In addition, eachbox was equipped with a small “house” light, a small speaker, and ametal grid floor. Forty-five-milligram dustless precision pellets (stan-dard chow; Bio-Serv, Flemington, NJ) were used as reward.

Animals (male Lister-Hooded rats, aged approximately 18 monthsand weighing approximately 350–450 g at the time of testing; HarlanLaboratories, Horst, The Netherlands) received treatment withina counter-balanced design (0, 1, 3, and 10 mg/kg JNJ-55511118,

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suspended in 0.5%HPMC, 10ml/kg p.o., 120minutes prior to testing).During testing, the rats experienced 15 trials at four different delays(1, 10, 20, and 30 seconds) per session. A session ended once animalscompleted all 60 trials or after 45 minutes. A trial started with theillumination of themagazine, followed by a nose poke at themagazine,turning off the receptacle light, and starting the sample phase. One oftwo levers was extended in a pseudorandom fashion, and the rat had20 seconds to respond. This caused the lever to be retracted andstarted the delay phase (variable 1–30 seconds). Once the delay hadpassed, the rat had 10 seconds to respond at the receptacle, causingthe magazine light to go off and the two levers to be extended (choicephase). A response at the lever opposite the sample lever within alimited hold of 10 secondswas counted as a correct response, and led tothe retraction of the levers, magazine illumination, a short tone, anddelivery of a food pellet. Pellet collection turned the magazine light offand started a 5-second intertrial interval. A response to the same leverthat was presented during the sample phase was counted as anincorrect response and resulted in the retraction of the responselevers, a short “time out” (10 seconds) in darkness. A lack of responseduring the sample or choice phases is counted as an omitted trial.

Percentage correct served as a measure of working memory. Therelative number of omitted trials and various response latenciesserved as measures of responsivity. Delayed nonmatch to position(DNMTP) percentage correct data were fitted to repeated-measureslogistic regression mixed-effect models, latencies were analyzedusing mixed-effect models, and percentage of errors of omissionwas determined by a logistic regression model. Main treatmenteffects were then analyzed by ANOVA followed by post-hoccontrasts.

ResultsJNJ-55511118 and JNJ-56022486 were discovered through

directed medicinal chemistry following a high-throughputscreening campaign targeting AMPA receptors containingTARP-g8. The structures of these molecules are shown inFig. 1.Calcium Flux. JNJ-55511118 and JNJ-56022486, along

with the nonselective AMPAR inhibitors CP-465022 andGYKI-53655, were evaluated for their ability to inhibitglutamate-evoked calcium flux in HEK-293 cells heterologouslyexpressing GluA subunits with and without TARPs. Inhibi-tion as a function of concentration for these compounds inassays using various combinations of human GluA and TARPsubunits is shown in Fig. 2. The fitted values for the potency ofinhibition at each target, averaged overmultiple experiments,are summarized in Table 1. JNJ-55511118 and JNJ-56022486potently inhibited every tested GluAx subunit, providedTARP-g8 was present. Both compounds showed little inhibi-tory activity up to the highest concentrations tested at TARP-less AMPARs and at AMPARs coexpressed with other TARPsor with cornichon family AMPA receptor auxiliary protein2 (CNIH2). In contrast, the noncompetitive inhibitors CP-465022 and GYKI-53655 showed no selectivity among AMPAreceptor subtypes, or among the TARP-containing AMPARs.

To explore the molecular basis of the selectivity of thesecompounds, we constructed chimeric proteins of TARP-g8 and-g4. Functional interaction with the AMPA receptor wasestablished by determining the potency of inhibition ofGluA1ocoexpressed with the chimeric TARPs. Figure 3A shows thetopology and nomenclature of the constructs used in theseexperiments. Figure 3C shows the change in potency ofinhibition for each compound as compared with the potencyat GluA1o-g8. In the first group of chimeras, we interchangedEX1 and the CT of TARP-g8 and -g4. Neither exchange alteredthe potency or efficacy of the compounds: chimeras 448444444and 444444448 (g4 with only EX1 or CT replaced) wereinsensitive, whereas 884888888 and 888888884 (g8 with onlyEX1 or CT replaced) were potently inhibited.In the next set of chimeras (888888884–844444444), we

progressively replaced sections of g8 with the correspondingregions of g4, starting from the C terminus. JNJ-55511118and JNJ-56022486 both lost their ability to inhibit theresponse beginning with chimera 888888844, implicatingTM4 in the functional activity of the compounds. Indeed,chimera 888888848 was completely insensitive to inhibition.The inverse chimera 444444484 was sensitive to these com-pounds, with a reduced potency.In chimeras 488888888–444444488, we progressively

replaced domains of TARP-g8, starting from the N terminus.The potency of JNJ-55511118 and JNJ-56022486 was un-changed in chimeras 488888888–444448888. However, whenTM3 was replaced (444444888), the compounds lost approx-imately 10-fold in potency. This suggests that TM3 is alsoinvolved in the functional activity of the compounds, althoughsomewhat lower in magnitude than TM4.We aligned the sequences for the human TARPs to identify

candidate residues that could account for the pharmacology ofthese compounds (Fig. 3B). TM4 contains four amino acidsunique to TARP-g8, and TM3 contains three. We generatedpoint mutations of g8, in each case mutating individualresidues to the corresponding one from g4. Figure 3D showsthe change in potency of inhibition for GluA1o coexpressedwith each of these constructs. Two of these point mutationsshowed altered potency of the TARP-selective compounds:G210A and V177I. G210A completely abolished activity of thecompounds, whereas V177I caused a 10-fold loss of potency.Double mutations of g4 and g2 in the corresponding locationsto the g8 residues conferred sensitivity of those TARPs toinhibition by JNJ-55511118 and JNJ-56022486.To identify additional residues which may be involved with

the functional activity, we scanned TARP-g8 in the vicinityG210 andV177.Wemade single-point alaninemutations fromN173 through G176 in TM3, and G209 through I214 in TM4.Most of these point mutations, when coexpressed withGluA1o, did not alter the potency of the g8-selective inhibitors.N173A caused a complete loss of inhibitory efficacy, whereasthe compounds were 10- to 100-fold less potent with G209Aand F213A.Selectivity. The selectivity of JNJ-55511118 and JNJ-

56022486 was evaluated at 1 mM against a panel of 52receptors, ion channels, and transporters using radioliganddisplacement assays (Cerep S.A., Poitiers, France). Data aresummarized in Supplemental Table 9. The compounds showedless than 50% binding at all tested targets, except for activity ofJNJ-55511118 at the serotonin receptor 2B (78% effect)and JNJ-56022486 at the melatonin receptor (57% effect).

Fig. 1. Chemical structures of JNJ-55511118, JNJ-56022486, and [3H]JNJ-56022486.


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JNJ-55511118 was further evaluated in a cell-based functionalassay using the recombinant human serotonin receptor 2B,and was determined to be an antagonist at this receptor, withan IC50 of 6 mM (data not shown). Thus, both compounds werea minimum of 100-fold selective against all tested targets.Electrophysiology. The AMPAR modulation of JNJ-

55511118 was explored in further detail using electrophysio-logical recordings. Figure 4A shows representative traces ofthe glutamate-evoked responses of cells expressing AMPAreceptors. In outside-out patches of Chinese hamster ovary

(CHO) cells expressing GluA1o-g8, a saturating concentrationof JNJ-55511118 produced a partial inhibition. Peak currentswith 1mMJNJ-55511118were reduced to 57.26 2.0% (mean6S.E.M.; N 5 7) relative to currents in the same patches prior toaddition of the compound. In contrast, peak currents inpatches of CHO cells expressing GluA1o-g2 were virtuallyunaffected by the presence of 1 mM JNJ-55511118 (97.6 62.0%, N 5 8).Weused cultured cerebellar granule cells fromneonatalmouse

as our model system for native AMPA receptors expressing

Fig. 2. Inhibition of glutamate-evoked calcium flux as functions of test compound concentration. The GluA subunit and the TARP are designated in thecaptions. A dash between the GluA and the TARP indicates that a fusion construct was used. A plus indicates that the GluA and TARP plasmids werecotransfected. Data points are the means of 2–42 data points from 1–22 individual experiments. (A–D) Concentration-response curves for the humanconstructs. (E and F) Concentration-response curves for nonhuman constructs. In each case, the GluA and the TARP subunits were both from thedesignated mammal.

TABLE 1Potency for inhibition of glutamate-evoked responses in calcium flux assaysData are represented as the mean pIC50 6 standard deviation of multiple measurements. The numbers in parenthesesindicate the number of measurements performed. Unless otherwise stated, all constructs are human.

GYKI-53655 CP-465022 JNJ-55511118 JNJ-56022486

GluA1o-g8 5.65 6 0.46 (14) 6.57 6 0.24 (20) 8.33 6 0.28 (22) 8.02 6 0.35 (10)GluA1o+ g8 5.72 6 0.42 (2) 6.26 6 0.25 (7) 7.95 6 0.21 (7) 7.79 6 0.22 (6)rat(GluA1o- g8) 5.00 6 0.41 (2) 6.18 6 0.37 (7) 7.89 6 0.3 (7) 7.45 6 0.41 (5)mouse(GluA1o- g8) 5.19 6 0.24 (2) 6.03 6 0.23 (8) 7.87 6 0.31 (7) 7.53 6 0.44 (5)monkey(GluA1o+ g8) 5.81 6 0.66 (2) 6.28 6 0.25 (4) 7.88 6 0.15 (7) 7.65 6 0.21 (6)dog(GluA1o+g8) 5.63 6 0.38 (2) 6.16 6 0.16 (4) 7.82 6 0.28 (7) 7.53 6 0.27 (6)GluA1i+g8 5.39 6 0.46 (2) 6.02 6 0.17 (8) 7.91 6 0.39 (7) 7.58 6 0.33 (5)GluA2i+g8 5.25 6–(1) 6.06 6 0.22 (6) 8.13 6 0.35 (2) 7.5 6 0.11 (2)GluA3o+g8 5.24 6 0.09 (2) 6.15 6 0.3 (9) 7.42 6 0.38 (6) 7.03 6 0.1 (4)GluA4o+g8 5.8 6 0 (2) 6.28 6 0.46 (2) 7.8 6 0.26 (4) 7.49 6 0.11 (4)GluA1o+g8+CNIH2 5.54 6 0.5 (2) 6.18 6 0.52 (12) 7.87 6 0.31 (7) 7.66 6 0.28 (4)GluA1i 5.11 6 0.29 (2) 5.94 6 0.15 (4) .5 (6) .5 (4)GluA1o+CNIH2 4.96 6 0.12 (2) 5.93 6 0.27 (7) .5 (6) .5 (4)GluA1o+g2 6.03 6 0.11 (2) 6.55 6 0.38 (5) .5 (5) .5 (4)GluA1o+g3 6.18 6 0.3 (2) 6.72 6 0.23 (8) .5 (6) .5 (4)GluA1o+g4 5.85 6 0.35 (2) 6.45 6 0.46 (8) .5 (6) .5 (4)GluA1o+g7 5.84 6 0.19 (2) 6.55 6 0.34 (5) .5 (6) .5 (3)

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TARP-g2, and acutely dissociated hippocampal neurons fromadult mouse for native AMPA receptors expressing TARP-g8.Figure 4A shows representative traces of the glutamate-evokedresponses of outside-out patches from cultured cerebellar neu-rons and whole-cell currents from acute hippocampal neurons.Analogous to the results with heterologously expressed AMPAreceptors, 1 mM JNJ-55511118 reduced peak glutamate-evokedcurrents in hippocampal neurons to 60.76 2.6% (N5 6) relativeto control, with virtually no effect on cerebellar currents (98.561.7%,N5 7). Supplemental Fig. 4 shows a summary of the effectof the glutamate-evoked peak current by JNJ-55511118 in a

variety of cell types; no inhibition was seen in cells expressingGluA1o, GluA1o-g2, GluA1i1g2, cultured cerebellar granulecells, or hippocampal neurons from TARP-g8 knockout mice.Figure 4B shows the glutamate-evoked peak current as a

function of concentration of JNJ-55511118 in outside-outpatches from CHO cells expressing human GluA1o-g8 andfrom acutely dissociated mouse hippocampal neurons. In bothcell types, the peak currents were partially inhibited at asaturating concentration. By nonlinear least-squares fitting toa logistic function with slope fixed to unity, the maximalinhibitionwas 55.86 1.9%, and themidpointwas 3.86 1.0 nM for

Fig. 3. Determination of location of specificity for TARP-selective compounds. (A) Schematic diagram indicating the sections of the proteins used forrepresentative chimeric TARPs (not drawn to scale). Transmembrane segments are depicted as wider lines. Segments from the different TARPs arecolor-coded: blue, g8; red, g4; green, g2. The chimeras are labeled with a nine-digit number; each digit indicates the TARP used for that section of theprotein, starting from the N terminus (NT). The diagrams for all of the chimeras are shown in Supplemental Fig. 3. (B) Sequence alignment of the humanisoforms of the TARPs in the TM3–TM4 regions. Vertical lines mark the predicted positions of the transmembrane domain regions. Highlights indicatepositions for which TARP-g8 is different from TARP-g4. (C and D) Potency of inhibition of the glutamate response of GluA1o coexpressed with each of thechimeras and point mutations. Potency is expressed as DpIC50: the difference between pIC50 of GluA1o coexpressed with the construct and pIC50 ofGluA1o+g8. (D) g8.DM is g8.G210A.V177I, g4.DM is g4.A189G.I156V, and g2.DM is g2.A184G.I153V. 55511118, JNJ-55511118; 56022486, JNJ-56022486; CP, CP-465022; GYKI, GYKI-53655; IN1, intracellular domain 1.


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GluA1o-g8. For hippocampal neurons, the maximal inhibitionwas 60.1 6 3.4%, and the midpoint was 15.0 6 4.6 nM.To assess the activity and selectivity of JNJ-55511118

during synaptic transmission, we recorded field excitatorypostsynaptic potentials (fEPSPs) from the CA1 region ofhippocampal slices from wild-type and TARP-g8 knockoutanimals (Fig. 4C). We did not observe an effect of JNJ-55511118 (1 mM) on the fEPSPs in CA1 of the hippocampusfrom TARP-g8 knockout animals (105.3 6 6.9% of baseline;N5 5), whereas the fEPSPs were inhibited in slices fromwild-type littermates (63.4 6 11.0% of baseline; N 5 6). We usedwhole-cell patch clamp in pyramidal CA1 neurons from

hippocampal slices to test the activity of JNJ-55511118(1 mM) on synaptic AMPAR currents. Electrical stimulation ofSchaffer collaterals evokedAMPAR-mediatedEPSCswhichwereinhibited by JNJ-55511118 to 73.0 6 4.0% of control (N 5 10;P 5 0.001 by paired-sample Student’s t test) (SupplementalFig. 5A). This effect appeared to be specific to postsynapticAMPA receptors since we did not observe changes in thepaired-pulse ratio orNMDAEPSCs (1236 16% of control;N510; P5 0.59 by paired-sample Student’s t test) (SupplementalFig. 5, C and D). We observed reduced synaptic summationwhen stimulated at 50 kHz in the presence of JNJ-55511118(Supplemental Fig. 5B).

Fig. 4. Electrophysiological evaluation of the effects of JNJ-55511118. (A) Outside-out recordings of glutamate-evoked currents in patches from cellsexpressing AMPA receptors, in the presence (red) and absence (black) of 1 mM JNJ-55511118. L-Glutamate (10 mM) was applied during the timedepicted by the gray bar. (B) Peak current evoked by 10 mM L-glutamate, normalized to control, in patches from cells (heterologous GluA1o-g8 oracutely dissociated mouse hippocampal pyramidal cells) as a function of concentration (mean 6 S.E.M.). (C) Bath application of JNJ-55511118partially inhibited the fEPSPs in CA1 hippocampal slices from wild-type mice, but not from TARP-g8 knockout (KO) mice. (D) Representativecurrents in outside-out patches from cells expressing GluA1i+g8. The middle panel shows the currents normalized to the peak, to visualize the fasterdesensitization in the presence of 1 mM JNJ-55511118 (red) or TARP-less GluA1i (blue). The lower panel is a box plot for desensitization timeconstants across conditions. The box plots show the median (line), mean (square), 25th and 75th percentiles (box), and minimum/maximum(whiskers). (E) Recovery from desensitization. Representative traces for recovery from desensitization in TARP-g8–containing AMPA receptors inthe absence (top) or presence of 1 mM JNJ-55511118 (middle). The lower panel shows the recovery from desensitization exponential fits for TARP-g8–containing AMPA receptors in the absence (black) or presence of JNJ-55511118 (red) and GluA1i-only expressing receptors (blue). (F, top panel)JNJ-55511118 (1 mM) inhibited peak currents evoked by a brief (1 ms) pulse of glutamate (10 mM). (F, middle panel) Currents normalized to peakshow the differences in current decay kinetics across conditions. (F, bottom panel) Summary bar graph for the deactivation time constants acrossconditions. (G) Expression of TARP-g8 (top-right panel) enhances the kainate (KA; 1 mM, normalized to 10 mM glutamate) efficacy compared withTARP-less AMPA receptors (top-left panel). JNJ-55511118 (1 mM) did not significantly affect the kainate/glutamate ratio in TARP-g8–containingAMPA receptors (bottom panels).

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To investigate the effect of JNJ-55511118 on desensitizationof g8-containing AMPA receptors, we used 500-ms applicationsof 10 mM L-glutamate on outside-out patches of HEK-293 cellsexpressing heterologous GluA1i1g8 (Fig. 4D). JNJ-55511118(1 mM) reduced peak currents to 63.8 6 4.1% of control andsteady-state currents to 25.4 6 3.7% of control (N 5 6). In thepresence of cyclothiazide, which removes desensitization,1 mM JNJ-55511118 inhibited glutamate-evoked currents to59.56 1.7% of control (N5 6; Supplemental Fig. 6). Inhibitionin the presence of cyclothiazide was not significantly differentfrom inhibition of peak currents (P 5 0.52), whereas it wassignificantly reduced compared with inhibition of the steady-state current (P , 0.001; one-way repeat measures ANOVAfollowed by Tukey’s test).In addition to reducing peak and steady-state currents, the

presence of 1 mM JNJ-55511118 accelerated the rate ofdesensitization, which can be observed by normalizing to thepeak currents (Fig. 4D, middle panel). After application of10 mM L-glutamate, GluA1i1g8 desensitized with a timeconstant of 7.96 0.8 ms (N5 6). In the presence of 1 mM JNJ-55511118, the time constant decreased to 5.7 6 0.5 ms (P ,0.001 by paired-sample Student’s t test). In the absence ofTARP, GluA1i desensitized considerably faster, with a timeconstant of 3.5 6 0.3 ms (N 5 5). We determined the recoveryfrom desensitization by measuring the peak current in re-sponse to paired applications of 10mML-glutamate, separatedby a variable delay (Fig. 4E). Recovery time constants weresimilar for GluA1i1g8 in the absence or presence of JNJ-55511118 (control tr,desens5 936 29ms,N5 7; JNJ-55511118tr,desens 1026 33 ms,N5 6; P5 1). Receptors in patches fromcells expressing TARP-less GluA1i recovered more slowlythan GluA1i1g8 (GluA1i tr,desens 5 163 6 10 ms, N 5 8; P 50.02 by Student’s t test).We investigated the effects of JNJ-55511118 on the de-

activation of TARP-g8–containing AMPA receptors. Fastapplication (1 ms) of glutamate to excised outside-out patchesgenerated rapidly deactivating currents. Figure 4F (top panel)shows that JNJ-55511118 partially inhibited the glutamate-evoked peak currents to 67 6 2% of control (N 5 9).Normalization of peak currents shows that JNJ-55511118increased the deactivation rate (Fig. 4F, middle panel). Afterapplication of 10 mM L-glutamate, GluA1i1g8 deactivatedwith a time constant of 4.66 0.9 ms (N5 9). In the presence of1 mM JNJ-55511118, the time constant decreased to 3.1 60.6 ms (P 5 0.001 by paired-sample Student’s t test). In theabsence of TARP, GluA1i deactivated considerably faster,with a time constant of 1.2 6 0.3 ms (N 5 6).To test for the possibility that JNJ-55511118may disrupt the

interaction between TARP g-8 and AMPA receptors, we lookedat the effects of JNJ-55511118 on kainate efficacy. TARPsenhance kainate efficacy for AMPA receptors (Tomita et al.,2005; Turetsky et al., 2005); the ratio of response to kainateversus glutamate is a sensitive assay for TARP/AMPARstoichiometry (Shi et al., 2009). As previously reported, TARP-lessAMPAreceptors hada considerably lower kainate/glutamateratio of steady-state currentswhen comparedwithg8-containingAMPARs (Fig. 4G, top panels). In the absence of TARP-g8, thekainate/glutamate ratio for GluA1i was 0.21 6 0.04 (N 5 4),whereas the kainate/glutamate ratio for GluA1i-g8 was 5.1 61.1 (N5 9). In the presence of JNJ-55511118 (Fig. 4G, bottom-left panel), the ratio was 4.2 6 0.7 (N 5 9). JNJ-55511118failed to significantly change the kainate/glutamate ratio of

the steady-state currents (P 5 0.58 by paired-sample Stu-dent’s t test); thus, the inhibition observedwith JNJ-55511118does not involve the dissociation of the TARP from the receptorcomplex.Binding Assays. We incorporated tritium into JNJ-

55511118 and JNJ-56022486 to explore their utility in radio-ligand binding assays. [3H]JNJ-56022486 proved to havelower nonspecific binding than [3H]JNJ-55511118, likely dueto its lower lipophilicity and higher free fraction in brain tissue(see Supplemental Table 10). Therefore, we focused on [3H]JNJ-56022486 for additional binding studies.Saturation binding in membranes from rat hippocampus

using [3H]JNJ-56022486 is shown in Fig. 5A. The fitted valueof the binding affinity was 27 6 3 nM, with Bmax 5 3.8 60.3 pmol/mg protein. In competition binding experiments(Fig. 5B), JNJ-55511118 and JNJ-56022486 fully displacedthe radioligand (20 nM) with Ki 5 26 6 7 and 19 6 6 nM,respectively (N 5 3). Neither Philanthotoxin-74 nor gluta-mate showed appreciable displacement of [3H]JNJ-56022486,whereas LY-395153 [a positive allosteric modulator (PAM)]and perampanel partially displaced the radioligand (Fig. 5B).Displacement by LY-395153 required the presence of 500 mMglutamate; the PAM did not displace the radioligand in theabsence of glutamate (data not shown).Autoradiograms showing total binding of [3H]JNJ-56022486

in brain slices from mouse, rat, and monkey are shown in Fig.5, D–H. An image from the Allen Brain Atlas of the expressionof CACNG8 in a corresponding coronal brain slice from anadult mouse (Lein et al., 2007) is shown in Fig. 5C. Theseimages indicate a high concentration of specific binding thatcorresponds well to the expression pattern of CACNG8.Pharmacokinetics. We determined the pharmacokinetic

and in vivo target occupancy profiles for JNJ-55511118 andJNJ-56022486. JNJ-55511118 achieved high plasma concen-trations upon oral (p.o.) and i.v. dosing (Fig. 6A). The compoundwas orally bioavailable in both species. In vivo clearance andvolume of distribution in rats were 4.8 ml/min/kg and 1.8 l/kg,respectively. Target occupancy was determined by ex vivoautoradiography of brain slices of animals dosed with testcompound, using [3H]JNJ-56022486 to probe for unoccupiedreceptors. JNJ-55511118 was highly brain-penetrant, andshowed high target occupancy upon oral dosing in both ratand mouse (Fig. 6, B and C). This compound also displayedlinear exposure as a function of dose (Fig. 6D). Although JNJ-56022486 also showed good oral bioavailability, the brainpenetration and target occupancy at 10 mg/kg were sub-stantially lower compared with JNJ-55511118 (SupplementalFig. 7). Therefore, we focused on JNJ-55511118 for addi-tional in vivo studies. In vitro measurements of tissuebinding of JNJ-55511118 showed 1.48 and 0.88% freefraction in rat and mouse plasma, respectively, and 0.24%free fraction in rat brain tissue. Supplemental Table 10shows a summary of the pharmacokinetic parameters de-rived for these two compounds.In Vivo Electrophysiology. For a more direct measure-

ment of the functional consequences of target engagement, weperformed in vivo electrophysiological recordings in thepyramidal cell layer of CA1 in the rat hippocampus, whilestimulating the Schaffer collateral projections from CA3.When dosed intravenously, JNJ-55511118 showed a rapid,dose-dependent inhibition of the evoked population spikeamplitude, with a fitted half-maximal effective dose (ED50)


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of 0.4 mg/kg (Fig. 7, A and B). One hour after dosing,recordings were terminated; plasma and brain tissue wereharvested for bioanalysis to measure the concentration ofJNJ-55511118. Fig. 7C shows the population spike inhibitionat 1 hour postdosing as a function of the measured brainconcentration for each animal. Superimposed in this graph isthe receptor occupancy as a function of brain concentration forrat from the experiments shown in Fig. 6E. The calculatedhalf-maximal effective concentration (EC50) values for thesetwo assays were quite similar (591 ng/ml, in vivo electrophys-iology; 545 ng/ml, autoradiography).EEG. To investigate the effect of functional g8-selective

inhibition on the cortical oscillations in freely behaving rats,sleep-wake architecture and EEG power spectral density wereevaluated after oral administration of JNJ-55511118 in a

dose-response experiment. A group of seven animals wereorally dosed at 2 hours into the light phase with vehicle (0.5%HPMC) or JNJ-55511118 (1, 3, and 10 mg/kg; 0.5% HPMCsuspension) in a randomized crossover design. EEG/EMGsignals and locomotor activity were recorded for up to 20hours after each pharmacological treatment; here, we showthe effects during the first 8 hours after administration.Oral administration of JNJ-55511118 elicited a clear dose-

dependent decrease in EEG activity during the wake state inall frequency bands above 4 Hz (Fig. 7G). Specifically, a dose-related reduction of the power spectral density in the theta(4–10Hz) [F(3, 18)5 26.73,P, 0.001], alpha (10–15Hz) [F(3, 18)535.80, P , 0.001], and beta (15–30 Hz) [F(3, 18) 5 168.10, P ,0.001] oscillations was observed from the lowest dose tested.The power spectral density in the delta oscillations was

Fig. 5. Radioligand binding assays. (A) Saturation binding assay using [3H]JNJ-56022486. Data points are the mean and standard deviation of fourindividual measurements. Specific binding (blue) was calculated as the difference in the means of total (black) and nonspecific (red) binding. Solid linesare fits to the data. (B) Competition binding assays using 20 nM [3H]JNJ-56022486. Data are the mean of four measurements. Solid lines are fits to thedata. (C) Expression pattern of the CACNG8 gene by in situ hybridization in a coronal section of mouse brain, downloaded from the Allen Mouse BrainAtlas (Lein et al., 2007) (ª 2015 Allen Institute for Brain Science, http://mouse.brain-map.org/experiment/show/72108823). (D–H) Receptor density inbrain slices using [3H]JNJ-56022486. Scale bars in each image are 5 mm. The color scale on the right shows the image intensity from low (blue) to high(red) radiation counts. Mouse (D) and rat (E) coronal brain slices. Mouse (F), rat (G), and monkey (H) sagittal brain slices.

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minimally affected, and only a small but significant decreasewas revealed at a dose of 1 mg/kg. Consequently, thecontribution of the EEG delta activity over the total power(relative delta power) was enhanced, since the absolute valuesfor power spectra in all of the higher frequency bands weredecreased.The 10-mg/kg dose, which should have achieved approxi-

mately 90% target occupancy (see Fig. 6F), represents thenear-saturating drug effect. At this dose, power in the wakestate in the theta, alpha, and beta bandswas reduced to 81.261.0, 70.6 6 1.3, and 63.8 6 1.2%, respectively, relative tocontrol. This substantial reduction in EEG power was accom-panied by a significant increase in locomotor activity forapproximately 1 hour postdose (Fig. 7H). In addition, therewas a dose-dependent increase in the latency to REM andNREM sleep (Supplemental Fig. 8).JNJ-55511118 also produced dose-dependent decreases in

absolute EEG power during REM and NREM sleep states(Supplemental Fig. 9). During NREM sleep, EEG oscillationswere significantly reduced from the 1-mg/kg dose onward inall measured frequency bands (1–30 Hz). During REM sleep,delta (1–4 Hz), theta (4–10 Hz), and beta (15–30 Hz) EEGoscillations were significantly reduced from the dose of 1 mg/kgonward. In contrast, EEG oscillations in the sigma frequencyrange (10–15 Hz) were minimally affected.Anticonvulsant Assays. We explored the anticonvul-

sant profile of JNJ-55511118 using several in vivo models.Figure 7, D and E shows the dose-response relationship of theactivity in the corneal kindling and 6 Hz models, as well asthe rotarod test. Based on the target occupancy in mouseshown in Fig. 6F, target occupancy should be well above 90%at doses above 40 mg/kg. Any additional pharmacology abovethat dose would likely be due to off-target effects. In the

corneal kindling model, JNJ-55511118 provided near-complete seizure protection at and above 5 mg/kg (p.o., 1 hourpostdosing). Twenty-five out of 32 animals had Racine scoresof zero at doses of 5–40mg/kg, indicating the complete absenceof observable effects of the stimulus. In comparison, 8/8animals in the vehicle control group had Racine scores of 5(rearing and falling with forelimb clonus). The curve fits to thecorneal kindling data in Fig. 7, D and E indicate that ED50 53.7 mg/kg, with a brain concentration EC50 of 938 ng/ml.JNJ-55511118 showed partial protection in the 6 Hz

models. At doses of 10 mg/kg and above, projected to givetarget occupancies of 80% or greater, 12/32 (37.5%) animalswere protected in the 6 Hz 32 mA model, and 11/40 (27.5%)were protected in the 6 Hz 44 mA model. This compound wasalso tested in the MES model; at 40 mg/kg p.o., 50% of theanimals showed seizure protection (N 5 8; data not shown).The curve fits to the 6 Hz 32 mA data indicate that ED50 518.3 mg/kg, with a plasma concentration EC50 of 4644 ng/mland 76%maximumprotection. For the 6Hz 44mA test, ED5056.5 mg/kg, with a plasma concentration EC50 of 2533 ng/mland 22% maximum protection.The Metrazol (PTZ) test was performed at t5 0.5 hour after

dosing with 40 mg/kg JNJ-55511118. The compound showedstrong protection (Fig. 7F); the mean threshold for clonusincreased from 33.36 4.9 to 49.56 10.0mg/kg PTZ (P, 0.001,two-sample t test), and for twitch, from 30.7 6 4.6 to 42.4 67.5 mg/kg (P , 0.001, two-sample t test).JNJ-55511118 was also tested in the amygdala kindling

model in rats. This compound protected 11/12 rats fromseizure (2 hours postdose, 10 mg/kg p.o., 0.5% HPMC suspen-sion), compared with 1/12 rats protected with vehicle alone.The after-discharge duration, shown in Supplemental Fig. 10,was significantly reduced by JNJ-55511118 to 356 7 seconds,

Fig. 6. Pharmacokinetics and target occupancy for JNJ-55511118 in rat and mouse. (A) Plasma concentration as a function of time in Sprague-Dawleyrats following oral (5 mg/kg p.o.) or intravenous (1 mg/kg) administration. (B and C) Brain and plasma concentrations in rat and mouse after a 10-mg/kgp.o. dose. Target occupancy was determined by autoradiography of brain slices. (D) Dose linearity of plasma and brain concentrations after p.o. dosing inrat (t = 4 hours) and mouse (t = 1 hour). (E and F) Target occupancy as determined by autoradiography after p.o. dosing in rat (t = 4 hours) andmouse (t =1 hour). In all panels except (E), data are presented as the mean and standard deviation.


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compared with 92 6 12 seconds with vehicle alone (mean 6S.E.M., N 5 12; P 5 4 � 1024, two-sample t test).Effects in Models of Learning/Memory/Cognition. In-

hibition of AMPA receptors, particularly in the hippocampusand prefrontal cortical areas, might be expected to impact

hippocampal plasticity and learning/memory mechanismsimportant for performance on spatial working memory tasks.The Morris water maze (MWM) provides a key method to

investigate spatial learning and memory in rodents (D’Hoogeand De Deyn, 2001). Figure 8, A and B shows that vehicle-treated

Fig. 7. Effects of JNJ-55511118 on in vivo measures. (A–C) Evoked neurotransmission in hippocampus using in vivo electrophysiology. Populationspikes (PS) were recorded in the pyramidal cell layer of rat hippocampal CA1, evoked by stimulation of Schaffer collaterals in CA3. (C) Responseamplitudes, normalized to predose baseline amplitudes, plotted as a function of time following i.v. administration of drug. Each data point represents theaverage response from two to four animals per dosing group. Drug was administered at time t = 0. (B) Dose-response profile of population spikeamplitude, averaged over the last 5 minutes of each 60-minute recording period in (A). (C) Population spike amplitude (black squares) for each animal asa function of brain concentration, as measured immediately after the recording. Superimposed is the target occupancy (blue circles), measured in ratswith oral dosing, from the autoradiography experiments shown in Fig. 6. Curves in (B) and (C) indicate nonlinear least-squares fits to a Hill function.(D–F) Anticonvulsant assays in mouse. (D and E) Dose response in corneal kindling (black) and 6 Hz models (red and green). Seizure protection wascalculated as the fraction of animals with Racine seizure scores of 3 or lower (N = 8 mice per cohort). Also shown is the fraction of animals from the 6 Hztests with motor impairment as measured on the rotarod (blue). Lines represent fits to a Hill function. (D) Data plotted as a function of dose. (E) Samedata as in (D), plotted using the average measured brain concentration in a parallel cohort of animals. (F) Protection in Metrazol (PTZ) test. Thethresholds for the amount of PTZ delivered to achieve twitch and clonus in mice (N = 10 per cohort) were significantly increased after dosing with JNJ-55511118 (40 mg/kg suspension, p.o.). The box plot shows the median (line), 25th and 75th percentiles (box), minimum/maximum (whiskers), andindividual data for each animal (circles). (G) EEG power activity during the wake state in rats. Power spectral density in the delta (1–4 Hz), theta (4–10Hz),alpha (10–15 Hz), and beta (15–30 Hz) bands were determined for the 8-hour period after compound or vehicle administration. Data are represented asmeans 6 S.E.M. of the same seven animals per dose. *P , 0.05, **P , 0.01, and ***P , 0.001 versus vehicle, based on one-way ANOVA followed byDunnett’s multiple-comparison post-hoc test. (H) Locomotor activity in rats. Activity counts were determined per 5-minute interval for the 120-minuteperiod after compound or vehicle administration. Data are presented as means 6 S.E.M. of the same seven animals per dose.

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animals obtained a typical learning curve during the 4training days evidenced by shortening of latency times (Fig.8A) and path length (Supplemental Fig. 11) to reach theplatform. Based upon the occupancy as a function of doseshown in Fig. 6E, the dose range of 0.63, 2.5, and 10 mg/kg p.o.should have given target occupancies of 50–90%. Surprisingly,the performance was not severely impacted; at the 0.63- and2.5-mg/kg doses during the training days, there was nostatistically significant deviation from performance ofvehicle-treated animals, apart from a small transient butstatistically significant improved latency time to platform onday 2 (which was not confirmed in an independent repeatstudy; data not shown). The animals in the 10-mg/kg cohortshowed a modest deficit in learning the platform location(increased time and path length to locate the platform) on the

first (P5 0.05,Coxproportional hazardsmodel) and second (P50.04) training days; however, on the third and fourth trainingdays, their performance was indistinguishable from vehicle-treated animals.In the DNMTP test, there was an overall effect of dose (P ,

0.01), with JNJ-55511118 decreasing the percentage of correctresponses at the two highest doses tested (3 and 10mg/kg; P,0.05 andP, 0.01, respectively), although the effect was rathermoderate (approximately 5% reduction; Fig. 8C). This wasaccompanied by a small, but significant, reduction in respon-sivity, as indicated by an increase in errors of omission andresponse latencies (Supplemental Fig. 12). Although therewas no significant drug� delay interaction (P. 0.05), post-hoccontrasts were computed against the vehicle group for thepercentage of correct responses, showing significant effects of

Fig. 8. Effects of JNJ-55511118 in assays of learning and memory in rodents. (A and B) Behavioral measures in the Morris water maze test. (A)Acquisition performance for each training day, measured as the time to locate the submerged platform. Animals were dosed 2 hours before the start ofthe procedure, and each animal had three training periods over 4 consecutive days. Data points are the mean and S.E.M. for N = 12 animals. (B)Performance in the probe trial. Immediately after the acquisition trials were completed on day 4, the platform was removed. Data shown are the fractionof time that the animals spent in each quadrant of the maze, for the first 30 seconds after entering the water maze. The submerged platform hadoriginally been in quadrant A (QA). (C and D) Primary measures in the DNMTP assay (N = 10 animals per dose group). (C) Overall fraction of correctresponses. (D) Fraction of correct responses separated by delay time. (E and F) Behavioral measures for the V-maze (N = 12 animals per dose group). (E)Discrimination index for the preference of rats for the new versus the familiar arm of the maze. Statistics are from comparisons to the vehicle controlgroup. (F) Total distance moved during the habituation and test phases for each dose group. The statistics are from comparisons to the vehicle cohort ofeach phase.


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the two higher doses at the shortest delay (1 second), but not atlonger delays (Fig. 8D). All tested doses increased the percent-age of trials omitted, and the two highest doses tested induced asignificant increase on latency measures (see SupplementalFig. 12). Themagnitudes of these effects were generally modest(less than 0.5 second for most measures).The V-maze was used to study the effects of JNJ-55511118

on working memory in rodents. This paradigm exploits thenatural tendency of rodents to explore a novel, nonthreateningenvironment. The V-maze avoids the use of aversive testconditions, such as electric shocks or deprivation, that mayhave nonspecific influences on the responses, and does notrequire prior learning of a rule.Vehicle-treated animals show a clear preference for the new

arm over the familiar arm they explored just before (discrim-ination index5 0.406 0.06; Fig. 8E). A partial impairment ofthis parameter is found in the 10-mg/kg treatment group (P50.0055, ANOVA). Concomitantly, a small increase in distancemoved is seen in a dose-dependent manner in the habituationphase (P , 0.0001) and at a dose of 10 mg/kg only in the testphase (P 5 0.0075; Fig. 8F).

DiscussionAMPA receptor signaling is a critical component of normal

and pathophysiological excitatory neuronal function, and hasbeen an attractive pharmacological target for both positiveand negative modulation. To date, all negative modulators ofAMPA receptors target the pore-forming GluA subunits.Although the presence of TARPs can modify potency andefficacy of modulators (Cokic and Stein, 2008), no publishedreports have shown any selectivity of inhibitors among theTARPs. Here, we report a novel pharmacological approach tonegatively modulate AMPA receptor signaling, with mole-cules selective for TARP-g8.JNJ-55511118 and JNJ-56022486 are highly potent, and

are exquisitely selective for AMPA receptors containingTARP-g8, with no detectable functional effect on TARP-lessAMPA receptors or those containing TARP-g2, -g3, -g4, or -g7.The data presented here suggest that these compounds exerttheir effects through a novel mechanism of action, via partialdisruption of a protein-protein interaction. JNJ-55511118exhibits excellent pharmacokinetics and brain penetration,and achieves high-target occupancy upon oral and intra-venous dosing. Tritiated JNJ-56022486 is useful as a radio-ligand for competitive binding studies, and for ex vivoautoradiography to determine target occupancy.TARP Selectivity. The g8–g4 chimeras were designed to

locate the site of selectivity. Previous studies have demon-strated that the TARP C terminus and EX1 domains havestrong effects on trafficking and functional interaction withthe GluA subunits (Tomita et al., 2005; Turetsky et al., 2005;Cais et al., 2014). Thus, a drug interacting at one of these tworegions could differentially impact the function of the AMPAreceptor complex. Chimera pairs (448444444, 884888888) and(444444448, 888888884) directly probed this question. Asshown in Fig. 3C, JNJ-55511118 and JNJ-56022486 inhibited888888884 (g8 with the C terminus from g4) and failed toinhibit 444444448 (g4 with the C terminus from g8). This pairof results suggests that the C terminus is not involvedwith thepharmacological activity of these two compounds. The chi-mera pair (448444444, 884888888) showed a similar failure to

alter the pharmacology of the backbone TARP, again suggest-ing that domain EX1 is not involved with the pharmacologicalactivity. Instead, domain scanning and direct substitutionsindicated that TM3 and TM4 govern the selectivity.The point-mutation studies allowed us to determine that

selectivity is entirely determined by two amino acids predictedto lie within TM3 and TM4. These two amino acids (G210 andV177) are unique to TARP-g8. Altering these two amino acidsto their corresponding components of TARP-g4 completelyabolished the potency of the compounds, whereas the TARP-g8 versions of these same two amino acids added to bothTARP-g2 and TARP-g4 confer identical sensitivity of theseTARPs to the compounds. According to the predicted topologyof the TARP (Fig. 3A), G210 and V177 are both 2–3 residuesdeep within the outer membrane surface.Data and alignments were retrieved from the UniProt

database (UniProt Consortium, 2015) for genes identified asCACNG8, from a selection of species. Sequence alignments ofTM3 and TM4 for the TARP-g8 in selected species are shownin Supplemental Fig. 13; these regions are highly conservedacross species. Curiously, the two key amino acids thatdetermine selectivity against TARP-g2 and -g4 (G210A andV177I) are also conserved across species (Supplemental Fig.14). Indeed, we found no species differences in potency orselectivity in rat, mouse, dog, monkey, or human isoforms(Fig. 2, E and F; Table 1). These two key amino acid changesare relatively small; each involves adding a single methylgroup in making the wild-type TARP-g8 protein completelyinsensitive to JNJ-55511118. In addition to these two keyresidues, alanine scanning indicated that N173 in TM4 andG209 and F213 in TM3 influence the potency of the com-pounds. These amino acids are adjacent to, or one alpha helixturn away from, the key selectivity locations. These fiveamino acids may form or contribute to the binding site forthese TARP modulators.Mechanism of Action. The radioligand binding assays

showed that JNJ-55511118 and JNJ-56022486 fully displaced[3H]JNJ-56022486, with affinities comparable to the potenciesas measured in the calcium flux assays. In contrast, ligandsthat bind to the agonist site (glutamate) and the pore(Philanthotoxin-74) showed no displacement. Ligands thatbind to the GluA PAM site (LY-395153) and the noncompet-itive antagonist site (perampanel), which couple allostericallywhen TARPs are associated with the AMPA receptor (Schoberet al., 2011), partially displaced [3H]JNJ-56022486. Partialdisplacement implies allosteric coupling between these sitesand the binding site for the TARP-g8 modulators; furtherstudies are required to investigate this phenomenon.The electrophysiology experiments allowed detailed char-

acterization of the effects of JNJ-55511118 upon the AMPAreceptor currents. The results are consistent with a partialdisruption of the interaction between TARP-g8 and the pore-forming GluA subunits. Type I TARPs provide a positivemodulatory influence upon the AMPA receptor (Howe, 2015).The electrophysiological studies showed that JNJ-55511118reverses some, but not all, of the effects of the TARP on theAMPA receptor complex. A saturating concentration of JNJ-55511118 reduces peak responses by 36–44%, and steady-state currents by ∼75%. This reduction is consistent withmasking the increases in single-channel conductance ob-served in studies of single-channel kinetics of TARPed versusTARP-less AMPA receptors (Shelley et al., 2012; Zhang et al.,

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2014). This inhibition is accompanied by an increase in thedesensitization and deactivation kinetic rates.Other aspects of the inhibition by JNJ-55511118 indicate

that TARP-g8 remains associated with the receptor complex.First, the kainate/glutamate ratio does not change as would beexpected with changes in TARP stoichiometry (Shi et al., 2009).TARPs enhance kainate efficacy for AMPA receptors (Tomitaet al., 2005; Turetsky et al., 2005), and the kainate-to-glutamateresponse ratio is a sensitive assay for TARP/AMPAR stoichio-metry (Shi et al., 2009). As previously reported, TARP-lessAMPA receptors had a considerably lower kainate/glutamateratio of steady-state currents when compared with AMPAreceptors containing TARP-g8. JNJ-55511118 failed to signif-icantly change the kainate/glutamate ratio of the steady-statecurrents, strongly suggesting that the inhibition observedwith JNJ-55511118 does not involve the dissociation of theTARP from the receptor complex. Second, the deactivation anddesensitization kinetics observed are considerably faster inthe TARP-less receptors when compared with the TARP-g8–containing receptors in the presence of JNJ-55511118.Third, JNJ-55511118 does not affect the recovery from de-sensitization, whereas the TARP-containing AMPA receptorsrecover from desensitization faster than TARP-less AMPAreceptors (Priel et al., 2005). JNJ-55511118 may partiallyaffect the GluA-TARP interaction required for reduced de-sensitization and deactivation, leaving intact those interac-tions required for kainate efficacy. Alternatively, TARP-g8may induce a unique conformational state on AMPARsnot observed with other TARPs, enabling a binding site onAMPARs for the inhibitors to reduce open-channelprobability.These features suggest that JNJ-55511118 does not cause

the TARP to dissociate from the AMPA receptor complex, butinstead modifies the protein-protein interaction between theTARP and GluA subunits. In a simplified gating modelcontaining single open, resting, and desensitized states (Sunet al., 2002), these effects are consistent with destabilization ofthe open state by JNJ-55511118. Additional kinetic studiescomparing TARP-less AMPA receptors to those containingTARP-g8 with and without JNJ-55511118 should reveal moredetails of the mechanism of action. In addition, mutations ofTARP-g8 at the key amino acids described earlier may provideadditionalmechanistic insight and structure-activity relation-ships regarding the interaction between TARPs and the GluAsubunits.Electrophysiological measurements in acutely dissociated

hippocampal neurons indicate that the modulation by JNJ-55511118 closely recapitulates the behavior in heterologoussystems. This suggests that, consistentwith previous findings,native hippocampal AMPA receptors are highly TARPed(i.e., contain a high level of TARP proteins), primarily withTARP-g8. It also indicates that the presence of the additionalaccessory proteins in the native system does not substantiallyalter the functional impact of the compound or the bindingpocket.In addition to their modulatory effects on gating and

channel conductance, TARPs have multiple additional effectsupon AMPARs: they modify surface expression (Rouach et al.,2005; Tomita et al., 2005); alter the pharmacology of the PAM,agonist, and noncompetitive antagonist binding sites (Tomitaet al., 2005; Turetsky et al., 2005; Schober et al., 2011); andparticipate in anchoring AMPARs to the synaptic scaffolding

(Chen et al., 2000). Whether JNJ-55511118 impacts theseadditional interactions between TARPs andAMPARs remainsan open question. Indeed, this molecule may prove useful indetermining the structure-function relationships of thesediverse phenomena.Synaptic Transmission. We tested the activity of JNJ-

55511118 in hippocampal slices, a preparation in which thenative synaptic receptors are found in association with compo-nents of the postsynaptic densities and associated auxiliarysubunits. Here, we found that JNJ-55511118 inhibited thesynaptic responses in the hippocampal CA1 region from wild-type mice but not from TARP-g8 knockout littermates. Consis-tent with specificity of JNJ-55511118 on postsynaptic AMPAreceptors, the compound only inhibited the AMPA EPSCs fromCA1 pyramidal neurons, but not theNMDAEPSCs, and did notaffect the paired-pulse ratio. Taken together, these data suggestthat JNJ-55511118 does not affect the presynaptic glutamaterelease probability, but instead acts directly on postsynapticAMPA receptors containing TARP-g8. We also observed re-duced synaptic summation when stimulated at 50 kHz in thepresence of JNJ-55511118 (Supplemental Fig. 5), suggesting apotential indication of this compound in states of hyperactivehippocampal activity, such as seizures. These results confirmthe activity of JNJ-55511118 on native postsynaptic AMPAreceptors under basal and high-frequency synaptic stimulation,and set the stage for a physiologic role of this compound inhippocampal activity.In anesthetized rats, JNJ-55511118 caused near-complete

inhibition of population spikes in CA1 driven by Shaffercollateral stimulation. The concentration dependence ofpopulation spike inhibition closely matched the target occu-pancy. The magnitude of inhibition of the population spikeamplitude is somewhat surprising, given the partial inhibi-tion of AMPA receptor current in in vitro electrophysiology(Fig. 4, A and B) and of the fEPSP slope in ex vivo slices (Fig.4C). Partial inhibition of AMPA receptors to the extentobserved with JNJ-55511118 is apparently sufficient toreduce the synaptic drive below the spiking threshold of thepostsynaptic neurons.

TABLE 2Summary of results of behavioral experiments with JNJ-55511118

Experiment Result Notes

AnticonvulsantPTZ 2 Increased threshold to seizureCorneal kindling 22 Complete seizure protectionAmygdala kindling 22 Complete seizure protection6 Hz 32 mA 2 Incomplete protection6 Hz 44 mA 2 Incomplete protectionMES 0

General behaviorRotarod 0Basal motor activity + Transient hyperlocomotionSleep/wake cycle 2 Decreased sleep durationSedation 0

Learning/memoryWater maze 2 Minor decrease in learning rateV-maze 2 Minor decrease in performanceDNMTP 2 Minor decrease in performance

EEG oscillationsWake 2 Decrease in theta, alpha, betaNREM 2 Decrease in delta, theta, sigma, betaREM 2 Decrease in delta, theta, beta

0, no effect; + increased response; –, decreased response; —, strong decreasedresponse.


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Effects In Vivo. Table 2 summarizes the results of behav-ioral tests performed using a saturating dose of JNJ-55511118. Quantitative EEG analysis in freely behaving ratsduring the wake state revealed a dose-dependent decrease inthe power bands of theta, alpha, and beta activity. These dataindicate that JNJ-55511118 produced some degree of EEGslowing, starting at a dose corresponding to 60% receptoroccupancy. This effect is consistent with the anticonvulsantproperties of the compound. JNJ-55511118 gave strong pro-tection in the corneal kindling and PTZ models in mice, and inthe amygdala kindling model in rats. Efficacy in the cornealkindling model was dose-dependent, and roughly approxi-mated the target occupancy as a function of plasma concen-tration. The compound also showed partial protection in the6 Hz and MES tests in mice.Modulation of AMPA receptor signaling has long been

considered an attractive strategy for the treatment of epilepsy(Rogawski, 2011). AMPAR antagonists show strong anticon-vulsant activity in preclinical models, and the noncompetitiveAMPAR inhibitor perampanel was recently approved as anadjunctive treatment of partial-onset seizures (Ko et al.,2015). The efficacy of topiramate may be mediated in part bymodulation of phosphorylation of neuronal AMPA receptors.As with all known anticonvulsant medications, both of thesedrugs exhibit dose-limiting side-effect profiles that limit theirclinical utility (Perucca and Gilliam, 2012). Modulation ofAMPAR signaling using a TARP-g8–selective mechanism hastwo distinct advantages that may result in an improvedtherapeutic margin. First, the expression of TARP-g8 withinthe brain indicates that the drug will have its largest effectwithin the hippocampus, while avoiding direct inhibitoryeffects on brain regions involved with motor coordinationandwakefulness. Second, the compounds negativelymodulatebut do not completely inhibit AMPAR signaling. This in-herently limits the maximal effect of the drug.AMPA receptors have been associated with regulation of

hippocampal plasticity and short-term memory mechanismsimportant for performance on spatial and working memorytasks. GluA1 knockout mice show impaired short-term habit-uation to recently experienced stimuli, which has beensuggested to affect performance in hippocampus-dependentspatial working memory tasks (Sanderson et al., 2009;Sanderson andBannerman, 2012). Hippocampal lesions affectlearning and memory in both human (Manns et al., 2003) andnonhuman species (Morris et al., 1982). Similarly, knockout ofTARP-g8 reduces hippocampal AMPA receptor function andsynaptic plasticity (Rouach et al., 2005; Fukaya et al., 2006),although the impact of learning, memory, and cognition inthese transgenic animals has not yet been reported.We addressed this issue by testing JNJ-55511118 in rats

using the delayed nonmatch to position, V-maze, and Morriswater maze tests. Performance in MWM is severely attenu-ated by manipulations that negatively impact hippocampalsignaling (Morris et al., 1982; Riedel et al., 1999). DNMTP isgenerally thought to be dependent upon hippocampal function(Steckler et al., 1998a), with degradation in performancefollowing hippocampal lesions (Chudasama and Muir, 1997;Winters and Dunnett, 2004) and direct intrahippocampalinfusion of drugs affecting cholinergic, GABAergic, or gluta-matergic function (Robinson andMao, 1997; Mao and Robinson,1998). Moreover, numerous studies have found that DNMTPis sensitive to systemic glutamatergicmanipulations (Steckler

et al., 1998b; Smith et al., 2011), although effects of AMPAreceptor blockade in the delayed match to position test, aclosely related paradigm to DNMTP, has been reported tobe more subtle than effects of NMDA receptor blockade(Stephens and Cole, 1996).In the MWM, animals in the highest-dose cohort showed

attenuated learning within training days 1 and 2 (as evi-denced by nearly flat learning curves), but it is remarkablethat the latency time during the first trial on the second andthird training day is close to that of the vehicle group on thelast trial of the day before, suggesting that the animalsconsolidate memories during the nights between the trainingsessions. This is consistent with absence of treatment effectson performance during the probe trial: all treatment groupsshowed indistinguishable preference for the area in the poolwhere the platform was located during the training days.JNJ-55511118 showed impairment in V-maze, MWM, andDNMTP. However, compared with effects following systemicadministration of the NMDA receptor antagonists dizocilpineor phencyclidine (Willmore et al., 2001), or the muscarinicantagonist scopolamine (Chudasama and Muir, 1997), theimpairment induced by JNJ-55511118 was relatively small.Our data are in line with those reported for GluA1 knockoutmice, which also show relatively mild learning and memoryimpairments compared with animals with hippocampal le-sions (Sanderson and Bannerman, 2012).Even at saturating doses, JNJ-55511118 showed a benign

side-effect profile, with no loss of motor coordination orsedation (Fig. 7; Supplemental Fig. 8). Overall behavior ofanimals dosed with JNJ-55511118 appeared largely normal,with only transient hyperlocomotion immediately after dosingand upon transfer into a novel environment. Considering therobust anticonvulsant profile, the strong inhibition of EEGsignals, and the expression of the target within the hippo-campus and cortex, the relatively mild impact upon learningand memory in the Morris water maze, DNMTP, and V-mazeassays is quite surprising. Thus, TARP-g8 inhibition withmolecules such as JNJ-55511118 shows strong potentialclinical utility as an anticonvulsant, particularly for thoseforms of epilepsy with a strong hippocampal component, suchas temporal lobe epilepsy.In summary, TARP-g8 modulators represent a novel phar-

macological class of molecules which possess an unprecedentedmechanism of action: partial disruption of a protein-proteininteraction between the pore-forming GluA subunit of AMPAreceptors and the TARP-g8 accessory protein. These com-pounds provide important tools at several levels: 1) themolecular pharmacology of this interaction, 2) dissection of thestructure-function relationship of the GluA-TARP interaction,and 3) the in vivo behavioral and therapeutic potential ofpartial inhibition of hippocampal excitability. Neuropsychiatricdisorders can be viewed as pathologic disruption of theexcitation/inhibition balance of specific structures, circuits, orsets of neurons. TARP-g8 modulators have the potential to betransformational anticonvulsants, particularly for medialtemporal lobe epilepsy. By avoiding the midbrain and hind-brain, AMPA receptor modulators selective for TARP-g8 mayattenuate seizures without side effects such as ataxia andsedation that are often seen with less-selective anticonvul-sants. Other clinical applications for this mechanism includeschizophrenia, particularly in the early stages where exces-sive limbic activity has been observed (Schobel et al., 2013),

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and anxiety disorders, given the reciprocal relationship be-tween hippocampal function and the effects of anxiolytic drugs(Gray and McNaughton, 2003).The existence of this mechanism of action has the potential

to greatly expand the number of druggable targets. There areat least 30 additional proteins in the AMPAR proteome(Schwenk et al., 2012), each with unique expression profilesand functional impact upon the AMPA receptor complex.Thus, just as TARP-g8 shows tissue specificity for thehippocampus, it may be possible to tune the effects of a drugtargeting other subunits for specific brain regions or neuronalsubtypes. Beyond AMPA receptors, most ion channels, andindeed drug targets from other protein classes, associate withaccessory proteins, some of which show expression patternsmore specific than the pore-forming subunit.

Acknowledgments

The authors acknowledge the contributions of the following indi-viduals associated with Janssen Research and Development for theircontributions to the previously described experiments. Ning Qincloned several GluA and TARP constructs. Raymond Rynberg de-veloped formulations for the in vivo studies. Nancy Aerts and JohnTalpos performed the DNMTP experiments. Steven Sutton managedthe back-crossing of the transgenic mice. Sofie Embrechts performedthe water maze and V-maze experiments. Tom Van de Casteeleperformed the statistical analysis of the behavioral data fromlearning/memory assays. Caroline Lanigan advised on statisticalanalyses. Leslie Nguyen, Minerva Batugo, and Brian Scott performedthe bioanalytical studies. The authors also thank the followingindividuals associated with NeuroAdjuvants, Inc. for performing theanticonvulsant experiments: H. Steve White, University of Utah(study oversight and consultation, study reporting); Cameron S.Metcalf, NeuroAdjuvants, Inc. (6 Hz, MES, corneal kindling, amyg-dala kindling, study design and management, data analysis andreporting); Misty D. Smith (study review/quality control, tissuecollection); Timothy Pruess, University of Utah (i.v. Metrazol test);Fabiola Vanegas, University of Utah (novel object and open fieldassays); Jenny Huff, University of Utah (amygdala kindling); CarlosH. Rueda, University of Utah (surgical amygdala electrode implanta-tion). Staff members at BioDuro, LLC (Beijing, China) performed thepharmacokinetic studies in rats and mice.

Authorship Contributions

Participated in research design: Maher, Ameriks, Savall, Liu,Dugovic, Wickenden, Carruthers, Lovenberg.

Conducted experiments: Maher, Wu, Ravula, Liu, Lord, Wyatt,Matta, Dugovic, Yun.

Contributed new reagents or analytic tools: Ravula.Performed data analysis: Maher, Wu, Liu, Lord, Wyatt, Matta,

Dugovic, Ver Donck, Steckler.Wrote or contributed to the writing of the manuscript: Maher,

Ameriks, Liu, Lord, Wyatt, Matta, Dugovic, Ver Donck, Steckler.

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JPET #231712

Discovery and characterization of AMPA receptor modulators

selective for TARP-8

Michael P. Maher, Nyantsz Wu, Suchitra Ravula, Michael K. Ameriks, Brad M. Savall, Changlu Liu,

Brian Lord, Ryan M. Wyatt, Jose Matta, Christine Dugovic, Sujin Yun, Luc Ver Donck, Thomas Steckler,

Alan D. Wickenden, Nicholas I. Carruthers, Timothy W. Lovenberg

Journal of Pharmacology and Experimental Therapeutics

Supplemental Materials

Maher et al., TARP-8-selective AMPAR modulators, J. Pharmacol. Exp. Ther. JPET #231712

2

Supplemental Tables

Supplemental Table 1. Primers, cloning sites, and vectors used in generating mammalian expression vectors for the wild-type genes used in this study. Cloning sites (highlighted in shaded letters) were introduced into primers to facilitate the cloning process.

Genes Forward primer (5’ to 3’) Reverse primer (5’ to 3’) Cloning sites Vector

Human GluA1o

ATGTCAGAATTCATGCAGCACATTTTTGCCTTCTTCTGCAC

TCGTCAGCGGCCGCGCAGCTTCGGGGGCTCTGTGAGTTGCGACA

EcoR1/Not1 pCIneo

Monkey GluA1o

AGTCTAGGATCCGCCACCATGCAGCACATTTTTGCCTTCTTCTGCACC

CTGTCAGCGGCCGCTTACAATCCCGTGGCTCCCAAGGGCATCCCTGAAC

BamH1/Not1 pcDNA4/TO

Dog GluA1o

AGTCATGGATCCGCCACCATGCAGCACATTTTTGCCTTCTTCTGCACC

ATGTCTGCGGCCGCTTACAATCCCGTGGCTCCCAAGGGCATCC


Mouse GluA1o

ATCTCAAAGCTTGCCACCATGCCGTACATCTTTGCCTTTTTCTGCAC

GTCTCAGCGGCCGCTTACAATCCTGTGGCTCCCAAGGGCATCC

HindIII/Not1 pcDNA4/TO

Rat GluA1o TCGTCAAAGCTTGCCACCATGCCGTACATCTTTGCCTTTTTCTGCACCGGTT

ACGTCTGCGGCCGCTTACAATCCTGTGGCTCCCAAGGGCATC


Human GluA1i

ATGTCAGAATTCATGCAGCACATTTTTGCCTTCTTCTGCAC

TCGTCAGCGGCCGCGCAGCTTCGGGGGCTCTGTGAGTTGCGACA

EcoR1/Not1 pcDNA4/TO

Human GluA2o

AGTCATAAGCTTGCCACCATGCAAAAGATTATGCATATTTCTGTCCT

ATGTCAGGCCGCCTAAATTTTAACACTTTCGATGCCATATAC


Human GluA3o

ATGTCAGGTACCGCCACCATGGCCAGGCAGAAGAAAATGGGGCAAA

ATGTCTGCGGCCGCACTAGATCTTAACACTCTCTGTTCCATAC


Human GluA4o

ATGTCTAAGCTTGCCACCATGAGGATTATTTCCAGACAGATTGTCTTGTTAT

AGTCTCGCGGCCGCTCTAAATTTTAATACTTTCGGTTCCATATACGT


Human CACNG2

AGTCGTGGATCCGCCACCATGGGGCTGTTTGATCGAGGTGTTC

TCTACTGCGGCCGCTTATACGGGGGTGGTCCGGCGGTTGGCTGT

BamH1/Not1 pcDNA3.1(+)

Human CACNG3

ATGTCAGAATTCATGAGGATGTGTGACAGAGGTATC

ATGTCAGCGGCCGCTCAGACGGGCGTGGTGCGCCTGTTGGCCGGATT

EcoR1/Not1 pcDNA3.1/ hygro(+)

Human CACNG4

ATGTCTGGATCCGCCACCATGGTGCGATGCGACCGCGGGCTGCAG

CTGTCAGCGGCCGCTCACACAGGGGTCGTCCGTCGGTTCAGCATGC


Human CACNG7

GTCATCGGATCCGCCACCATGAGTCACTGCAGCAGCCGCGCCCTGA

CTGTACGCGGCCGCTCAGCAGGGCGAGGTGGAGATGTGCAGGTG


Human CACNG8

ATGTCAGAATTCCATGGAGTCGCTGAAGCGCTGGAACGAAGA

GTCATCGCGGCCGCCTACACAGGCGTGGTTTTCCTGTTGAGCGTGTT

EcoR1/Not1 pcDNA3.1(+)

Mouse CACNG8

AGTCTAGAATTCCAACAGCAGCAACAACAGCAACAGCAACAAGCCACCATGGAGTCATTGAAACGCTGGAATGA

AGTCTAGCGGCCGCCTACACGGGCGTGGTTTTCCTGTTGAGC


Rat CACNG8

AGTCTAGAATTCCAACAGCAGCAACAACAGCAACAGCAACAAGCCACCATGGAATCATTGAAACGCTGGAATGAAG

CTGTCAGCGGCCGCCTACACGGGCGTGGTTTTCCTGTTGAGCGTG

EcoR1/Not1 pcDNA3.1

Monkey CACNG8

CTGTACGAATTCGCCACCATGGAGTCGCTGAAGCGCTGGAACGA

GTCTACGCGGCCGCCTACACAGGCGTGGTTTTCCTGTTGAGCGTGTTG


Dog CACNG8

CTGTCAGAATTCGCCACCATGGAGTCGCTGAAGCGCTGGAACGA

CTGTCAGCGGCCGCCTACACTGGAGTAGTTTTTCTGTTGAGA

EcoR1/Not1 pcDNA3.1

Human CNIH2

GTCTACGGATCCGCCACCATGGCGTTCACCTTCGCCGCGTTCTGCT

CTGTCAGCGGCCGCTTAGAAACTCACCAACGTATAAACCATACT



3

Supplemental Table 2. Primers, cloning sites, and vectors used to generate expression vectors for fusions of GluA1o and TARP-8 used in this study. Shaded sequences in primers are restriction site introduced to facilitate the cloning process.

Primers and templates for PCR to generate GluA1-FLOP fusion protein expression constructs 5’ end PCR 3’ end PCR Full length PCR Forward

primer Reverse primer

Template Forward primer

Reverse primer


Reverse primer

Template

Human GluA1o-CACNG8

P1 P2 Human GluA1o

P3 P4 Human CACNG8 (codon optimized)*

P1 P4 5’end + 3’ end

Mouse GluA1o-CACNG8

P5 P6 Mouse GluA1o

P7 P8 Mouse CACNG8

P5 P8 5’end + 3’ end

Rat GluA1o-CACNG8

P9 P10 Rat GluA1o P11 P12 Rat CACNG8

P9 P12 5’end + 3’ end

Primer sequences (5’ to 3’)

P1 ATGTCAGAATTCATGCAGCACATTTTTGCCTTCTTCTGCAC P2 CTGCTGCTGCTGCTGCAGTCCTGTTGCTCCCAGAGGCATCCCGCTTGAATGGCTCATACATGGAATCGATTGCATGGACTTGGGGAA

GTC P3 AGCAACAGGACTGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGGAGTTCGCAACTATGGAGAGCCTGAAGCGGTGGAACGAGGAAC P4 ACGTCAGCGGCCGCTCAGACGGGTGTGGTTTTTCGGTTCAGTGTA P5 ATCTCAAAGCTTGCCACCATGCCGTACATCTTTGCCTTTTTCTGCAC P6 TTGTTGCTGTTGCTGTTGTTGCTGCTGTTGGAATTCCAATCCTGTGGCTCCCAAGGGCATCCCT P7 CAGCAGCAACAACAGCAACAGCAACAAGCCACCATGGAGTCATTGAAACGCTGGAATGAAG P8 AGTCTAGCGGCCGCCTACACGGGCGTGGTTTTCCTGTTGAGC P9 TCGTCAAAGCTTGCCACCATGCCGTACATCTTTGCCTTTTTCTGCACCGGTT P10 GGCTTGTTGCTGTTGCTGTTGTTGCTGCTGTTGGAATTCCAATCCTGTGGCTCCCAAGGGCATCC P11 AGCAGCAACAACAGCAACAGCAACAAGCCACCATGGAATCATTGAAACGCTGGAATGAAGAG P12 CTGTCAGCGGCCGCCTACACGGGCGTGGTTTTCCTGTTGAGCGTG


4

Supplemental Table 3. Coding sequences and predicted amino acid sequences for fusion proteins comprising human, mouse, and rat gluA1o and CACNG8.

Construct DNA Sequence Amino acid sequence Human GluA1o-CACNG8

ATGCAGCACATTTTTGCCTTCTTCTGCACCGGTTTCCTAGGCGCGGTAGTAGGTGCCAATTTCCCCAACAATATCCAGATCGGGGGATTATTTCCAAACCAGCAGTCACAGGAACATGCTGCTTTTAGATTTGCTTTGTCGCAACTCACAGAGCCCCCGAAGCTGCTCCCCCAGATTGATATTGTGAACATCAGCGACAGCTTTGAGATGACCTATAGATTCTGTTCCCAGTTCTCCAAAGGAGTCTATGCCATCTTTGGGTTTTATGAACGTAGGACTGTCAACATGCTGACCTCCTTTTGTGGGGCCCTCCACGTCTGCTTCATTACGCCGAGCTTTCCCGTTGATACATCCAATCAGTTTGTCCTTCAGCTGCGCCCTGAACTGCAGGATGCCCTCATCAGCATCATTGACCATTACAAGTGGCAGAAATTTGTCTACATTTATGATGCCGACCGGGGCTTATCCGTCCTGCAGAAAGTCCTGGATACAGCTGCTGAGAAGAACTGGCAGGTGACAGCAGTCAACATTTTGACAACCACAGAGGAGGGATACCGGATGCTCTTTCAGGACCTGGAGAAGAAAAAGGAGCGGCTGGTGGTGGTGGACTGTGAATCAGAACGCCTCAATGCTATCTTGGGCCAGATTATAAAGCTAGAGAAGAATGGCATCGGCTACCACTACATTCTTGCAAATCTGGGCTTCATGGACATTGACTTAAACAAATTCAAGGAGAGTGGCGCCAATGTGACAGGTTTCCAGCTGGTGAACTACACAGACACTATTCCGGCCAAGATCATGCAGCAGTGGAAGAATAGTGATGCTCGAGACCACACACGGGTGGACTGGAAGAGACCCAAGTACACCTCTGCGCTCACCTACGATGGGGTGAAGGTGATGGCTGAGGCTTTCCAGAGCCTGCGGAGGCAGAGAATTGATATATCTCGCCGGGGGAATGCTGGGGATTGTCTGGCTAACCCAGCTGTTCCCTGGGGCCAAGGGATCGACATCCAGAGAGCTCTGCAGCAGGTGCGATTTGAAGGTTTAACAGGAAACGTGCAGTTTAATGAGAAAGGACGCCGGACCAACTACACGCTCCACGTGATTGAAATGAAACATGACGGCATCCGAAAGATTGGTTACTGGAATGAAGATGATAAGTTTGTCCCTGCAGCCACCGATGCCCAAGCTGGGGGCGATAATTCAAGTGTTCAGAACAGAACATACATCGTCACAACAATCCTAGAAGATCCTTATGTGATGCTCAAGAAGAACGCCAATCAGTTTGAGGGCAATGACCGTTACGAGGGCTACTGTGTAGAGCTGGCGGCAGAGATTGCCAAGCACGTGGGCTACTCCTACCGTCTGGAGATTGTCAGTGATGGAAAATACGGAGCCCGAGACCCTGACACGAAGGCCTGGAATGGCATGGTGGGAGAGCTGGTCTATGGAAGAGCAGATGTGGCTGTGGCTCCCTTAACTATCACTTTGGTCCGGGAAGAAGTTATAGATTTCTCCAAACCATTTATGAGTTTGGGGATCTCCATCATGATTAAAAAACCACAGAAATCCAAGCCGGGTGTCTTCTCCTTCCTTGATCCTTTGGCTTATGAGATTTGGATGTGCATTGTTTTTGCCTACATTGGAGTGAGTGTTGTCCTCTTCCTGGTCAGCCGCTTCAGTCCCTATGAATGGCACAGTGAAGAGTTTGAGGAAGGACGGGACCAGACAACCAGTGACCAGTCCAATGAGTTTGGGATATTCAACAGTTTGTGGTTCTCCCTGGGAGCCTTCATGCAGCAAGGATGTGACATTTCTCCCAGGTCCCTGTCTGGTCGCATCGTTGGTGGCGTCTGGTGGTTCTTCACCTTAATCATCATCTCCTCATATACAGCCAATCTGGCCGCCTTCCTGACCGTGGAGAGGATGGTGTCTCCCATTGAGAGTGCAGAGGACCTAGCGAAGCAGACAGAAATTGCCTACGGGACGCTGGAAGCAGGATCCACTAAGGAGTTCTTCAGGAGGTCTAAAATTGCTGTGTTTGAGAAGATGTGGACATACATGAAGTCAGCAGAGCCATCAGTTTTTGTGCGGACCACAGAGGAGGGGATGATTCGAGTGAGGAAATCCAAAGGCAAATATGCCTACCTCCTGGAGTCCACCATGAATGAGTACATTGAGCAGCGGAAACCCTGTGACACCATGAAGGTGGGAGGTAACTTGGATTCCAAAGGCTATGGCATTGCAACACCCAAGGGGTCTGCCCTGAGAAATCCAGTAAACCTGGCAGTGTTAAAACTGAACGAGCAGGGGCTTTTGGACAAATTGAAAAACAAATGGTGGTACGACAAGGGCGAGTGCGGCAGCGGGGGAGGTGATTCCAAGGACAAGACAAGCGCTCTGAGCCTCAGCAATGTGGCAGGCGTGTTCTACATCCTGATCGGAGGACTTGGACTAGCCATGCTGGTTGCCTTAATCGAGTTCTGCTACAAATCCCGTAGTGAATCCAAGCGGATGAAGGGTTTTTGTTTGATCCCACAGCAATCCATCAACGAAGCCATACGGACATCGACCCTCCCCCGCAACAGCGGGGCAGGAGCCAGCAGCGGCGGCAGTGGAGAGAATGGTCGGGTGGTCAGCCATGACTTCCCCAAGTCCATGCAatcgatTCCATGTATGAGCCATTCAAGCGGGATGCCTCTGGGAGCAACAGGACTGcagcagcagcagcagcagcagcagcagcaggagttcgcaactATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MQHIFAFFCTGFLGAVVGANFPNNIQIGGLFPNQQSQEHAAFRFALSQLTEPPKLLPQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSFPVDTSNQFVLQLRPELQDALISIIDHYKWQKFVYIYDADRGLSVLQKVLDTAAEKNWQVTAVNILTTTEEGYRMLFQDLEKKKERLVVVDCESERLNAILGQIIKLEKNGIGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPAKIMQQWKNSDARDHTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVPWGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQAGGDNSSVQNRTYIVTTILEDPYVMLKKNANQFEGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDGKYGARDPDTKAWNGMVGELVYGRADVAVAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFSFLDPLAYEIWMCIVFAYIGVSVVLFLVSRFSPYEWHSEEFEEGRDQTTSDQSNEFGIFNSLWFSLGAFMQQGCDISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERMVSPIESAEDLAKQTEIAYGTLEAGSTKEFFRRSKIAVFEKMWTYMKSAEPSVFVRTTEEGMIRVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDSKGYGIATPKGSALRNPVNLAVLKLNEQGLLDKLKNKWWYDKGECGSGGGDSKDKTSALSLSNVAGVFYILIGGLGLAMLVALIEFCYKSRSESKRMKGFCLIPQQSINEAIRTSTLPRNSGAGASSGGSGENGRVVSHDFPKSMQSIPCMSHSSGMPLGATGLQQQQQQQQQQEFATMESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

Mouse GluA1o-CACNG8

ATGCCGTACATCTTTGCCTTTTTCTGCACCGGTTTTCTAGGTGCGGTTGTGGGTGCCAATTTCCCCAACAATATCCAGATAGGGGGATTATTTCCAAACCAACAATCACAGGAACATGCGGCTTTTAGGTTTGCTTTGTCACAACTCACGGAGCCCCCCAAGCTGCTTCCCCAGATCGATATTGTGAACATCAGCGACAGCTTTGAGATGACTTACCGATTCTGTTCCCAGTTCTCCAAAGGAGTGTACGCCATCTTTGGATTTTATGAACGAAGGACTGTCAACATGCTGACCTCCTTCTGTGGGGCCCTCCATGTGTGCTTCATCACTCCAAGTTTTCCCGTTGACACATCCAATCAGTTTGTCCTTCAGCTGCGCCCGGAACTACAGGAAGCTCTCATTAGCATTATCGACCATTACAAGTGGCAGACTTTTGTCTACATTTATGATGCTGACCGGGGCCTGTCAGTCCTGCAGAGAGTCTTGGATACAGCCGCCGAGAAGAACTGGCAGGTGACGGCTGTCAACATTCTAACAACCACGGAGGAAGGATACCGGATGCTCTTTCAGGACCTGGAGAAGAAAAAGGAGAGGCTGGTGGTGGTGGACTGTGAATCAGAACGCCTCAACGCCATCCTGGGCCAGATTGTGAAGCTAGAAAAGAACGGCATCGGGTACCACTACATCCTCGCCAACCTGGGCTTCATGGACATTGACTTAAATAAGTTCAAGGAGAGTGGAGCCAATGTGACAGGTTTCCAACTGGTGAACTACACAGACACGATCCCAGCCAGAATCATGCAGCAGTGGAGGACAAGTGACGCTCGAGACCACACACGGGTGGACTGGAAGAGACCCAAGTACACCTCTGCGCTCACCTACGATGGGGTGAAGGTGATGGCTGAGGCTTTCCAGAGCCTGCGGAGGCAGAGAATTGATATATCTCGCCGGGGGAATGCTGGGGATTGTCTGGCTAACCCAGCTGTTCCCTGGGGCCAAGGGATCGACATCCAGAGAGCTCTGCAGCAGGTGCGATTTGAAGGTTTAACAGGAAACGTGCAGTTTAATGAGAAAGGACGCCGGACCAACTACACGCTCCACGTGATTGAAATGAAACATGACGGCATCCGAAAGATTGGTTACTGGAATGAAGATGATAAGTTTGTCCCTGCAGCCACCGATGCCCAAGCTGGGGGCGATAATTCAAGTGTTCAGAACAGAACATACATCGTCACAACAATCCTAGAAGATCCTTATGTGATGCTCAAGAAGAACGCCAATCAGTTTGAGGGCAATGACCGTTACGAGGGCTACTGTGTAGAGCTGGCGGCAGAGATTGCCAAGCACGTGGGCTACTCCTACCGTCTGGAGATTGTCAGTGATGGAAAATACGGAGCCCGAGACCCTGACACGAAGGCCTGGAATGGCATGGTGGGAGAGCTGGTCTATGGAAGAGCAGATGTGGCTGTGGCTCCCTTAACTATCACTTTGGTCCGGGAAGAAGTTATAGATTTCTCCAAACCATTTATGAGTTTGGGGATCTCCATCATGATTAAAAAACCACAGAAATCCAAGCCGGGTGTCTTCTCCTTCCTTGATCCTTTGGCTTATGAGATTTGGATGTGCATTGTTTTTGCCTACATTGGAGTGAGTGTTGTCCTCTTCCTGGTCAGCCGCTTCAGTCCCTATGAATGGCACAGTGAAGAGTTTGAGGAAGGACGGGACCAGACAACCAGTGACCAGTCCAATGAGTTTGGGATATTCAACAGTTTGTGGTTCTCCCTGGGAGCCTTCATGCAGCAAGGATGTGACATTTCTCCCAGGTCCCTGTCTGGTCGCATCGTTGGTGGCGTCTGGTGGTTCTTCACCTTAATCATCATCTCCTCATATACAGCCAATCTGGCCGCCTTCCTGACCGTGGAGAGGATGGTGTCTCCCATTGAGAGTGCAGAGGACCTAGCGAAGCAGACAGAAATTGCCTACGGGACGCTGGAAGCAGGATCCACTAAGGAGTTCTTCAGGAGGTCTAAAATTGCTGTGTTTGAGAAGATGTGGACATACATGAAGTCAGCAGAGCCATCAGTTTTTGTGCGGACCACAGAGGAGGGGATGATTCGAGTGAGGAAATCCAAAGGCAAATATGCCTACCTCCTGGAGTCCACCATGAATGAGTACATTGAGCAGCGGAAACCCTGTGACACCATGAAGGTGGGAGGTAACTTGGATTCCAAAGGCTATGGCATTGCAACACCCAAGGGGTCTGCCCTGAGAAATCCAGTAAACCTGGCAGTGTTAAAACTGAACGAGCAGGGGCTTTTGGACAAATTGAAAAACAAATGGTGGTACGACAAGGGCGAGTGCGGCAGCGGGGGAGGTGATTCCAAGGACAAGACAAGCGCTCTGAGCCTCAGCAATGTGGCAGGCGTGTTCTACATCCTGATCGGAGGACTTGGACTAGCCATGCTGGTTGCCTTAATCGAGTTCTGCTACAAATCCCGTAGTGAATCCAAGCGGATGAAGGGTTTTTGTTTGATCCCACAGCAATCCATCAACGAAGCCATACGGACATCGACCCTCCCCCGCAACAGCGGGGCAGGAGCCAGCGGAGGAAGTGGCAGTGGAGAGAATGGCAGAGTGGTCAGCCAGGACTTCCCCAAGTCCATGCAATCCATTCCCTGCATGAGCCACAGTTCAGGGATGCCCTTGGGAGCCACAGGATTGGAATTCCAACAGCAGCAACAACAGCAACAGCAACAAGCCACCATGGAGTCATTGAAACGCTGGAATGAAGAGAGGGGTTTGTGGTGTGAAAAGGGCGTTCAGGTACTACTGACCACCATCGGCGCCTTCTCGGCTTTTGGCCTCATGACCATCGCCATCAGCACTGACTACTGGCTCTACACAAGAGCTCTCATCTGCAACACCACCAACCTCACAGCAGGTGATGACGGACCACCCCATCGTGGGGGCAGTGGCTCCTCCGAGAAGAAGGACCCTGGGGGCCTCACACATTCAGGCCTCTGGCGGATATGCTGCCTGGAAGGGTTGAAAAGAGGTGTCTGCGTGAAGATCAACCACTTCCCGGAGGACACGGACTACGACCACGACAGCGCGGAGTACCTGCTCCGAGTAGTCCGGGCTTCCAGCATCTTTCCTATCCTGAGCGCCATCCTGCTGCTGCTCGGGGGCGTGTGCGTAGCTGCCTCCCGCGTCTACAAGTCCAAAAGGAACATCATCCTGGGCGCAGGGATCCTGTTCGTGGCAGCAGGCTTGAGCAACATCATCGGGGTGATTGTGTACATATCGGCCAACGCCGGCGAGCCTGGCCCCAAGAGGGACGAGGAGAAGAAAAACCACTACTCGTACGGCTGGTCCTTCTACTTCGGCGGGCTGTCCTTCATCCTGGCTGAGGTGATCGGAGTACTGGCCGTCAACATCTACATCGAGCGCAGCCGCGAGGCACACTGCCAATCACGCTCGGACCTGCTCAAGGCCGGCGGCGGCGCGGGCGGCAGTGGCGGGAGCGGCCCCTCGGCCATCCTCCGTCTGCCCAGTTACCGCTTCCGCTACCGCCGCCGCTCCCGCTCCAGCTCCCGAGGCTCCAGCGAGGCGTCGCCATCCCGGGATGCGTCTCCCGGCGGCCCCGGGGGCCCGGGCTTCGCCTCCACGGACATCTCCATGTACACGCTCAGCCGCGACCCGTCCAAGGGCAGCGTGGCTGCGGGGCTGGCGAGCGCCGGTGGCGGCGGCAGCGGTGCCGGCGTGGGTGCCTACGGCGGGGCGGCCGGGGCGGCGGGGGGCGGCGGGGCGGGCTCGGAGCGGGACCGCGGGAGCTCGGCGGGTTTTCTCACGCTGCACAACGCCTTCCCCAAGGAAGCGGCGTCCGGCGTCACGGTCACAGTCACCGGACCGCCCGCTGCACCCGCGCCCGCGCCCGCGCCGCCCGCTCCTGCAGCGCCCGCGCCCGGGACCCTGTCCAAAGAGGCCGCGGCGTCCAACACCAACACGCTCAACAGGAAAACCACGCCCGTGTAG

MPYIFAFFCTGFLGAVVGANFPNNIQIGGLFPNQQSQEHAAFRFALSQLTEPPKLLPQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSFPVDTSNQFVLQLRPELQEALISIIDHYKWQTFVYIYDADRGLSVLQRVLDTAAEKNWQVTAVNILTTTEEGYRMLFQDLEKKKERLVVVDCESERLNAILGQIVKLEKNGIGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPARIMQQWRTSDARDHTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVPWGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQAGGDNSSVQNRTYIVTTILEDPYVMLKKNANQFEGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDGKYGARDPDTKAWNGMVGELVYGRADVAVAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFSFLDPLAYEIWMCIVFAYIGVSVVLFLVSRFSPYEWHSEEFEEGRDQTTSDQSNEFGIFNSLWFSLGAFMQQGCDISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERMVSPIESAEDLAKQTEIAYGTLEAGSTKEFFRRSKIAVFEKMWTYMKSAEPSVFVRTTEEGMIRVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDSKGYGIATPKGSALRNPVNLAVLKLNEQGLLDKLKNKWWYDKGECGSGGGDSKDKTSALSLSNVAGVFYILIGGLGLAMLVALIEFCYKSRSESKRMKGFCLIPQQSINEAIRTSTLPRNSGAGASGGSGSGENGRVVSQDFPKSMQSIPCMSHSSGMPLGATGLEFQQQQQQQQQQATMESLKRWNEERGLWCEKGVQVLLTTIGAFSAFGLMTIAISTDYWLYTRALICNTTNLTAGDDGPPHRGGSGSSEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRGSSEASPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLASAGGGGSGAGVGAYGGAAGAAGGGGAGSERDRGSSAGFLTLHNAFPKEAASGVTVTVTGPPAAPAPAPAPPAPAAPAPGTLSKEAAASNTNTLNRKTTPV


5

Rat GluA1o-CACNG8

ATGCCGTACATCTTTGCCTTTTTCTGCACCGGTTTTCTAGGTGCGGTTGTGGGTGCCAATTTCCCCAACAATATCCAGATAGGGGGATTATTTCCAAACCAACAATCACAGGAACATGCGGCTTTTAGGTTTGCTTTGTCACAACTCACGGAGCCCCCCAAGCTGCTTCCCCAGATCGATATTGTGAACATCAGCGACAGCTTTGAGATGACTTACCGATTCTGTTCCCAGTTCTCCAAAGGAGTGTACGCCATCTTTGGATTTTATGAACGAAGGACTGTCAACATGCTGACCTCCTTCTGTGGGGCCCTCCATGTGTGCTTCATCACTCCAAGTTTTCCCGTTGACACATCCAATCAGTTTGTCCTTCAGCTGCGCCCGGAACTACAGGAAGCTCTCATTAGCATTATCGACCATTACAAGTGGCAGACTTTTGTCTACATTTATGATGCTGACCGGGGCCTGTCAGTCCTGCAGAGAGTCTTGGATACAGCCGCCGAGAAGAACTGGCAGGTGACGGCTGTCAACATTCTAACAACCACGGAGGAAGGATACCGGATGCTCTTTCAGGACCTGGAGAAGAAAAAGGAGAGGCTGGTGGTGGTGGACTGTGAATCAGAACGCCTCAACGCCATCCTGGGCCAGATTGTGAAGCTAGAAAAGAACGGCATCGGGTACCACTACATCCTCGCCAACCTGGGCTTCATGGACATTGACTTAAATAAGTTCAAGGAGAGTGGAGCCAATGTGACAGGTTTCCAACTGGTGAACTACACAGACACGATCCCAGCCAGAATCATGCAGCAGTGGAGGACAAGTGACTCTCGAGACCACACACGGGTGGACTGGAAGAGACCCAAGTACACCTCTGCGCTCACCTACGATGGGGTGAAGGTGATGGCTGAGGCTTTCCAGAGCCTGCGGAGGCAGAGAATTGATATATCTCGCCGGGGGAATGCTGGGGATTGTCTGGCTAACCCAGCTGTTCCCTGGGGCCAAGGGATCGACATCCAGAGAGCTCTGCAGCAGGTGCGATTTGAAGGTTTAACAGGAAACGTGCAGTTTAATGAGAAAGGACGCCGGACCAACTACACGCTCCACGTGATTGAAATGAAACATGACGGCATCCGAAAGATTGGTTACTGGAATGAAGATGATAAGTTTGTCCCTGCAGCCACCGATGCCCAAGCTGGGGGCGATAATTCAAGTGTTCAGAACAGAACATACATCGTCACAACAATCCTAGAAGATCCTTATGTGATGCTCAAGAAGAACGCCAATCAGTTTGAGGGCAATGACCGTTACGAGGGCTACTGTGTAGAGCTGGCGGCAGAGATTGCCAAGCACGTGGGCTACTCCTACCGTCTGGAGATTGTCAGTGATGGAAAATACGGAGCCCGAGACCCTGACACGAAGGCCTGGAATGGCATGGTGGGAGAGCTGGTCTATGGAAGAGCAGATGTGGCTGTGGCTCCCTTAACTATCACTTTGGTCCGGGAAGAAGTTATAGATTTCTCCAAACCATTTATGAGTTTGGGGATCTCCATCATGATTAAAAAACCACAGAAATCCAAGCCGGGTGTCTTCTCCTTCCTTGATCCTTTGGCTTATGAGATTTGGATGTGCATTGTTTTTGCCTACATTGGAGTGAGTGTTGTCCTCTTCCTGGTCAGCCGCTTCAGTCCCTATGAATGGCACAGTGAAGAGTTTGAGGAAGGACGGGACCAGACAACCAGTGACCAGTCCAATGAGTTTGGGATATTCAACAGTTTGTGGTTCTCCCTGGGAGCCTTCATGCAGCAAGGATGTGACATTTCTCCCAGGTCCCTGTCTGGTCGCATCGTTGGTGGCGTCTGGTGGTTCTTCACCTTAATCATCATCTCCTCATATACAGCCAATCTGGCCGCCTTCCTGACCGTGGAGAGGATGGTGTCTCCCATTGAGAGTGCAGAGGACCTAGCGAAGCAGACAGAAATTGCCTACGGGACGCTGGAAGCAGGATCCACTAAGGAGTTCTTCAGGAGGTCTAAAATTGCTGTGTTTGAGAAGATGTGGACATACATGAAGTCAGCAGAGCCATCAGTTTTTGTGCGGACCACAGAGGAGGGGATGATTCGAGTGAGGAAATCCAAAGGCAAATATGCCTACCTCCTGGAGTCCACCATGAATGAGTACATTGAGCAGCGGAAACCCTGTGACACCATGAAGGTGGGAGGTAACTTGGATTCCAAAGGCTATGGCATTGCAACACCCAAGGGGTCTGCCCTGAGAAATCCAGTAAACCTGGCAGTGTTAAAACTGAACGAGCAGGGGCTTTTGGACAAATTGAAAAACAAATGGTGGTACGACAAGGGCGAGTGCGGCAGCGGGGGAGGTGATTCCAAGGACAAGACAAGCGCTCTGAGCCTCAGCAATGTGGCAGGCGTGTTCTACATCCTGATCGGAGGACTTGGACTAGCCATGCTGGTTGCCTTAATCGAGTTCTGCTACAAATCCCGTAGTGAATCCAAGCGGATGAAGGGTTTTTGTTTGATCCCACAGCAATCCATCAACGAAGCCATACGGACATCGACCCTCCCCCGCAACAGCGGGGCAGGAGCCAGCGGAGGAGGTGGCAGTGGAGAGAATGGCAGAGTGGTCAGCCAGGACTTCCCCAAGTCCATGCAATCCATTCCCTGCATGAGCCACAGTTCAGGGATGCCCTTGGGAGCCACAGGATTGGAATTCCAACAGCAGCAACAACAGCAACAGCAACAAGCCACCATGGAATCATTGAAACGCTGGAATGAAGAGAGGGGTTTGTGGTGCGAAAAGGGCGTTCAGGTACTACTGACCACCATAGGCGCCTTTGCAGCTTTTGGCCTCATGACCATCGCCATCAGCACTGACTACTGGCTCTACACAAGAGCTCTCATCTGCAACACCACCAACCTCACAGCAGGTGATGATGGACCACCCCATCGTGGGGGCAGTGGCTCCTCAGAGAAGAAGGACCCTGGGGGCCTCACACATTCAGGCCTCTGGCGGATATGCTGCCTGGAAGGGTTGAAAAGAGGTGTCTGCGTGAAGATCAACCACTTCCCGGAGGACACGGACTACGACCACGACAGCGCGGAGTACCTGCTCCGAGTAGTCCGGGCCTCCAGCATCTTTCCTATCCTGAGCGCCATCCTGCTGCTGCTCGGGGGCGTGTGCGTAGCTGCCTCTCGCGTCTACAAATCCAAAAGGAACATCATCCTGGGCGCAGGGATCCTGTTCGTGGCAGCAGGCCTGAGCAACATCATCGGGGTGATCGTGTACATATCGGCAAACGCGGGCGAGCCAGGCCCCAAGAGGGACGAGGAGAAGAAAAACCACTATTCGTATGGCTGGTCCTTCTACTTCGGCGGGCTGTCATTCATCCTGGCCGAGGTGATCGGCGTGCTAGCCGTCAACATCTACATCGAGCGCAGCCGCGAGGCACACTGCCAATCACGCTCGGACCTACTCAAGGCCGGCGGCGGCGCGGGCGGCAGTGGCGGGAGCGGCCCCTCGGCCATCCTCCGTCTGCCCAGTTACCGCTTCCGCTACCGCCGCCGCTCCCGCTCCAGCTCCCGAGGCTCCAGCGAGGCCTCGCCATCGCGGGATGCGTCTCCCGGCGGCCCCGGGGGCCCGGGCTTCGCCTCCACGGACATCTCCATGTACACGCTCAGTCGCGACCCGTCCAAGGGCAGCGTGGCTGCGGGGCTGGCGAGCGCCGGGGGTGGAGGCGGCGGTGCCGGCGTGGGTGCCTACGGCGGGGCGGCCGGGGCAGCGGGGGGCGGCGGGACGGGCTCGGAGCGGGACCGAGGGAGCTCAGCGGGCTTCCTCACGCTGCACAACGCCTTCCCCAAGGAGGCGGCGTCCGGCGTCACGGTCACGGTCACCGGACCGCCCGCTGCGCCGGCGCCCGCGCCGCCCGCTCCTGCAGCGCCCGCGCCCGGGACGCTGTCCAAAGAGGCCGCCGCGTCCAACACCAACACGCTCAACAGGAAAACCACGCCCGTGTAG

MPYIFAFFCTGFLGAVVGANFPNNIQIGGLFPNQQSQEHAAFRFALSQLTEPPKLLPQIDIVNISDSFEMTYRFCSQFSKGVYAIFGFYERRTVNMLTSFCGALHVCFITPSFPVDTSNQFVLQLRPELQEALISIIDHYKWQTFVYIYDADRGLSVLQRVLDTAAEKNWQVTAVNILTTTEEGYRMLFQDLEKKKERLVVVDCESERLNAILGQIVKLEKNGIGYHYILANLGFMDIDLNKFKESGANVTGFQLVNYTDTIPARIMQQWRTSDSRDHTRVDWKRPKYTSALTYDGVKVMAEAFQSLRRQRIDISRRGNAGDCLANPAVPWGQGIDIQRALQQVRFEGLTGNVQFNEKGRRTNYTLHVIEMKHDGIRKIGYWNEDDKFVPAATDAQAGGDNSSVQNRTYIVTTILEDPYVMLKKNANQFEGNDRYEGYCVELAAEIAKHVGYSYRLEIVSDGKYGARDPDTKAWNGMVGELVYGRADVAVAPLTITLVREEVIDFSKPFMSLGISIMIKKPQKSKPGVFSFLDPLAYEIWMCIVFAYIGVSVVLFLVSRFSPYEWHSEEFEEGRDQTTSDQSNEFGIFNSLWFSLGAFMQQGCDISPRSLSGRIVGGVWWFFTLIIISSYTANLAAFLTVERMVSPIESAEDLAKQTEIAYGTLEAGSTKEFFRRSKIAVFEKMWTYMKSAEPSVFVRTTEEGMIRVRKSKGKYAYLLESTMNEYIEQRKPCDTMKVGGNLDSKGYGIATPKGSALRNPVNLAVLKLNEQGLLDKLKNKWWYDKGECGSGGGDSKDKTSALSLSNVAGVFYILIGGLGLAMLVALIEFCYKSRSESKRMKGFCLIPQQSINEAIRTSTLPRNSGAGASGGGGSGENGRVVSQDFPKSMQSIPCMSHSSGMPLGATGLEFQQQQQQQQQQATMESLKRWNEERGLWCEKGVQVLLTTIGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGDDGPPHRGGSGSSEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRGSSEASPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLASAGGGGGGAGVGAYGGAAGAAGGGGTGSERDRGSSAGFLTLHNAFPKEAASGVTVTVTGPPAAPAPAPPAPAAPAPGTLSKEAAASNTNTLNRKTTPV


6

Supplemental Table 4. Location of splice points used to generate chimeras of human TARPs 8, 4, and 2. The numbering is based upon the amino acid sequence for each protein.

region start end start end start end

full length 1 425 1 327 1 323

NT 1 18 1 7 1 7

TM1 19 40 8 29 8 29

EX1 41 129 30 108 30 105

TM2 130 152 109 131 106 128

IN1 153 157 132 136 129 133

TM3 158 181 137 160 134 157

EX2 182 205 161 183 158 178

TM4 206 229 184 208 179 203

CT 230 425 209 327 204 323


7

Supplemental Table 5. Primers and templates used for PCR reactions to generate TARP chimeric protein expression constructs. The chimeras in this table are labelled using a shorthand notation (C1-C21 and D1); see Supplemental Table 6 for the translation for the shorthand notation to the nine-digit chimera name. Chimeras C1-C13 were codon-optimized and synthesized in a pD2610v12 vector by DNA 2.0 (http://dna20.com, Menlo Park CA). Chimeras C20, C21, and D1 were codon-optimized and synthesized in-house.

5’ end 3’ end Full length PCR Chi

mera Forward primer

Reverse primer


Reverse primer


Reverse primer

Template

C14 P21 P43 CACNG4 P44 P24 CACNG8 P21 P24 5’end + 3’ end






Primer sequences (5’ to 3’)

P21 ACGTCAGAATTCGCCACCATGCAATCGATTCCATGTATGAGC P22 AACCTCAACTCTTTATTTTTCTCAATATAGATATTGACGGCCAG P23 CTGGCCGTCAATATCTATATTGAGAAAAATAAAGAGTTGAGGTT P24 CTGTATCGGCCGCTCACACAGGGGTCGTCCGTCGGTTCAGCA P25 GAATTCGCCACCATGGTGCGATGCGACCGCGGGCTGCAGAT P26 GCAATGGGCTTCTCGACTGCGCTCAATGTAAATGTTTACAGCCA P27 TGGCTGTAAACATTTACATTGAGCGCAGTCGAGAAGCCCATTGC P28 ACGTCAGCGGCCGCTCAGACGGGTGTGGTTTTTCGGTTCAGT P29 ACAGAGCTCCAAAGTAAAAAGACCAGCCATAGGAGTAGTGGT P30 ACCACTACTCCTATGGCTGGTCTTTTTACTTTGGAGCTCTGT P31 GTCACCTGTGTTGCTGGAAATATAGACGATCACGCCAATGAT P32 ATCATTGGCGTGATCGTCTATATTTCCAGCAACACAGGTGAC P33 AAGAGGATGCCGGCACTGAGGACGATATTTCTTTTGGACTTGTACACCCGGCTTGCT

G P34 CAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCGTCCTCAGTGCCGGCATCCTCT

T P35 GAGGACGATGTTGTTCTTGCGGCTGTACACCCGGCTTGCTGCCACGCA P36 TGCGTGGCAGCAAGCCGGGTGTACAGCCGCAAGAACAACATCGTCCTC P37 TGGTGCTGAGGATGGGGAAGACGGAGCTAGCCCGGACCACTCGCA P38 TGCGAGTGGTCCGGGCTAGCTCCGTCTTCCCCATCCTCAGCACCA P39 GAGTACAGCCAGTAGTCGGTGGAAATAGCGATTGTCATCAGCCCAA P40 TTGGGCTGATGACAATCGCTATTTCCACCGACTACTGGCTGTACTC


8

P41 GTGGTCAGCAGCATCTGCAGTCCTTTTTCGCACCACAGCCCTC P42 GAGGGCTGTGGTGCGAAAAAGGACTGCAGATGCTGCTGACCAC P43 CCCACTGTGGTCAGCAGGACCTGCAGCCCGCGGTCGC P44 GCGACCGCGGGCTGCAGGTCCTGCTGACCACAGTGGG P45 GTATACAGCCAGTAGTCAGTGGAGATGGCGATGGCCATGAGCGAGA P46 TCTCGCTCATGGCCATCGCCATCTCCACTGACTACTGGCTGTATAC P47 CCAGAATGATATTTCTTTTGGACTTGTAGATCCTGCCAGCACCGATGC P48 GCATCGGTGCTGGCAGGATCTACAAGTCCAAAAGAAATATCATTCTGG P49 GCCGGCTCCCAGAATGATGTTGTTCTTGCGGCTGTAGA P50 TCTACAGCCGCAAGAACAACATCATTCTGGGAGCCGGC P51 GTCCGGGCTCCCCTGCATTGGCGCTAATGTAGACGATGATACCGATG P52 CATCGGTATCATCGTCTACATTAGCGCCAATGCAGGGGAGCCCGGAC P53 GATAAAAGACAGTCCCCCAAAGTAGAAAGACCAGCCGTAGTTGTAATGGTTCT P54 AGAACCATTACAACTACGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATC


9

Supplemental Table 6. The complete DNA coding regions and the protein sequences of the CACNG8/CACNG4 chimeras.

construct DNA Sequence protein sequence C1 (888888884)

ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGGCTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCGCACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGTGTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCCATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCATCCTTGGTGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAGTGATTGTGTACATCAGCGCAAACGCCGGAGAGCCCGGCCCGAAGCGCGATGAAGAAAAGAAGAACCATTACAGCTACGGCTGGTCCTTCTACTTCGGGGGTCTGTCGTTCATCTTGGCGGAAGTCATCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV..

C2 (444444448)

ATGGTCAGATGTGATAGAGGATTGCAAATGCTGCTCACTACCGCCGGGGCTTTCGCCGCCTTTTCACTCATGGCGATTGCCATCGGTACCGACTACTGGCTGTACTCCTCGGCCCACATCTGCAATGGCACTAACCTCACCATGGACGACGGACCCCCGCCACGCCGGGCTCGGGGCGACTTGACCCATTCGGGCCTGTGGAGGGTCTGCTGTATTGAGGGGATCTACAAGGGTCACTGCTTCCGGATTAACCACTTCCCTGAGGACAACGACTACGATCACGACTCCTCCGAGTATCTTCTGCGCATCGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGTGGACTGTGCATTGGGGCGGGCCGCATCTACTCCCGCAAAAACAACATCGTGCTTTCGGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCAGCAACACCGGAGATCCCTCAGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTCGGGGCACTGTCGTTCATCGTGGCGGAAACCGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAGGTCCAGAGAGGCACACTGCCAGTCCCGGAGCGACCTCCTGAAGGCCGGTGGAGGGGCCGGAGGCTCCGGAGGCAGCGGCCCATCGGCCATTCTGCGCCTGCCGAGCTACAGGTTTCGGTATCGGCGGCGCTCAAGATCATCCTCCCGCTCGTCCGAACCCTCCCCGAGCCGGGACGCCTCGCCCGGAGGACCGGGTGGCCCGGGATTCGCCTCCACCGACATCAGCATGTACACTCTGTCAAGAGATCCAAGCAAAGGAAGCGTCGCCGCGGGCCTCGCTGGCGCCGGAGGGGGCGGGGGTGGAGCAGTGGGAGCCTTCGGGGGCGCCGCTGGAGGAGCCGGGGGCGGTGGCGGAGGAGGCGGCGGAGCCGGTGCCGAGCGCGATCGGGGGGGCGCATCCGGTTTCCTGACGCTGCACAACGCGTTCCCGAAGGAAGCGGGCGGAGGAGTGACCGTGACTGTGACCGGACCGCCTGCCCCGCCTGCGCCTGCGCCCCCTGCTCCTTCCGCGCCTGCCCCCGGAACTCTCGCCAAGGAGGCCGCCGCCTCTAACACCAACACACTCAACCGAAAGACTACCCCCGTGTAGTGA

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGALSFIVAETVGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV..

C3 (888888844)

ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGGCTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCGCACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGTGTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCCATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCATCCTTGGTGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAGTGATTGTGTACATCAGCGCAAACGCCGGAGAGCCCGGCCCGAAGCGCGATGAAGAAAAGAAGAACCATTACAGCTACGGCTGGTCCTCCTTCTACTTCGGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV..

C4 (888888444)

ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGGCTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCGCACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGTGTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCCATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCATCCTTGGTGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAGTGATTGTGTACATCAGCTCCAACACGGGAGATCCCAGCGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTCGGGGCACTGTCGTTCATCGTGGCGGAAACCGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV..

C5 (888884444)

ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGGCTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCGCACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGTGTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCCATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCGTGCTTTCCGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTCGGGGCACTGTCGTTCATCGTGGCGGAAACCGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV..

C6 (888844444)

ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGGCTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCGCACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGTGTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCCATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACTCCCGGAAAAACAACATCGTGCTTTCCGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTCGGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV..

C7 (888444444)

ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGGCTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCGCACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGTGTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGTGGACTGTGCATTGGCGCGGGTCGCATCTACTCCCGGAAAAACAACATCGTGCTTTCCGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTCGGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV..


10

C8 (884444444)

ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGGCTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACTCAAGCGCCCACATCTGCAATGGCACTAACCTCACCATGGACGACGGACCTCCGCCTCGCCGGGCTAGGGGCGACTTGACCCATTCGGGCCTGTGGAGGGTCTGCTGTATCGAGGGGATCTACAAGGGTCACTGCTTCCGCATTAACCACTTCCCTGAGGACAACGACTACGATCACGACTCCTCCGAGTATCTTCTGCGCATTGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGTGGACTGTGCATTGGCGCGGGTCGCATCTACTCCCGGAAAAACAACATCGTGCTTTCCGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTCGGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV..

C9 (844444444)

ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGACTGCAAATGCTGCTCACTACCGCGGGGGCTTTCGCCGCCTTTTCCCTCATGGCGATTGCCATCGGCACCGACTACTGGCTGTACAGCAGCGCCCACATCTGCAATGGCACTAACCTCACCATGGACGACGGACCTCCGCCGCGCCGGGCTCGCGGCGACTTGACCCATTCGGGCCTGTGGAGGGTCTGCTGTATCGAGGGGATCTACAAGGGTCACTGCTTCCGGATTAACCACTTCCCTGAGGACAACGACTACGATCACGACTCCTCCGAGTATCTTCTGCGCATTGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGTGGACTGTGCATTGGCGCGGGTCGCATCTACTCCCGGAAAAACAACATCGTGCTTTCCGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTCGGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA

MESLKRWNEERGLWCEKGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV..

C10 (884888888)

ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGGCTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACTCCTCGGCCCACATCTGCAATGGCACTAACCTCACCATGGACGACGGACCCCCGCCACGCCGGGCTCGGGGCGACTTGACCCATTCGGGCCTGTGGAGGGTCTGCTGTATTGAGGGGATCTACAAGGGTCACTGCTTCCGGATTAACCACTTCCCTGAGGACAACGACTACGATCACGACTCCTCCGAGTATCTTCTGCGCATCGTGCGGGCCTCCTCGATTTTCCCCATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCATCCTTGGTGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAGTGATTGTGTACATCAGCGCAAACGCCGGAGAGCCCGGCCCGAAGCGCGATGAAGAAAAGAAGAACCATTACAGCTACGGCTGGTCCTTCTACTTCGGGGGCCTGTCGTTCATCCTGGCGGAAGTCATCGGAGTGCTTGCCGTGAATATCTACATCGAGAGGTCCAGAGAGGCACACTGCCAGTCCCGGAGCGACCTCCTGAAGGCCGGTGGAGGGGCCGGAGGCTCCGGAGGCAGCGGCCCATCGGCCATTCTGCGCCTGCCGAGCTACAGGTTTCGGTATCGGCGGCGCTCAAGATCATCCTCCCGCTCGTCCGAACCCTCCCCGAGCCGGGACGCCTCGCCCGGAGGACCGGGTGGCCCGGGATTCGCCTCCACCGACATCAGCATGTACACTCTGTCAAGAGATCCAAGCAAAGGAAGCGTCGCCGCGGGCCTCGCTGGCGCCGGAGGGGGCGGGGGTGGAGCAGTGGGAGCCTTCGGGGGCGCCGCTGGAGGAGCCGGGGGCGGTGGCGGAGGAGGCGGCGGAGCCGGTGCCGAGCGCGATCGGGGGGGCGCATCCGGTTTCCTGACGCTGCACAACGCGTTCCCGAAGGAAGCGGGCGGAGGAGTGACCGTGACTGTGACCGGACCGCCTGCCCCGCCTGCGCCTGCGCCCCCTGCTCCTTCCGCGCCTGCCCCCGGAACTCTCGCCAAGGAGGCCGCCGCCTCTAACACCAACACACTCAACCGAAAGACTACCCCCGTGTAGTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV..

C11 (448444444)

ATGGTCAGATGTGATAGAGGATTGCAAATGCTGCTCACTACCGCCGGGGCTTTCGCCGCCTTTTCCCTCATGGCGATTGCCATCGGTACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCGCACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGTGTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGTGGACTGTGCATTGGCGCGGGTCGCATCTACTCCCGGAAAAACAACATCGTGCTTTCCGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCTCGAACACAGGAGATCCCTCCGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTCCTTCTACTTCGGGGCTCTGTCGTTCATCGTGGCGGAAACGGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACCAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCTCCCCATATGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACCGAAGCGTCCCCGAGCCGGGACGTGTCGCCCATGGGACTCAAGATTACTGGAGCCATCCCTATGGGCGAACTCAGCATGTACACTCTGTCAAGAGAGCCACTCAAAGTGACCACTGCCGCCTCCTACTCGCCGGATCAGGAGGCATCCTTCCTGCAAGTCCACGATTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGACGCACTACCCCCGTGTAGTGA

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV..

C12 (444444484)

ATGGTCAGATGTGATAGAGGATTGCAAATGCTGCTCACTACCGCCGGGGCTTTCGCCGCCTTTTCACTCATGGCGATTGCCATCGGTACCGACTACTGGCTGTACTCCTCGGCCCACATCTGCAATGGCACTAACCTCACCATGGACGACGGACCCCCGCCACGCCGGGCTCGGGGCGACTTGACCCATTCGGGCCTGTGGAGGGTCTGCTGTATTGAGGGGATCTACAAGGGTCACTGCTTCCGGATTAACCACTTCCCTGAGGACAACGACTACGATCACGACTCCTCCGAGTATCTTCTGCGCATCGTGCGGGCCTCCTCGGTGTTCCCCATCCTGTCCACCATCCTGCTGCTCCTGGGTGGACTGTGCATTGGGGCGGGCCGCATCTACTCCCGCAAAAACAACATCGTGCTTTCGGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAATCATTGTGTACATCAGCAGCAACACCGGAGATCCCTCAGACAAGCGCGATGAAGATAAGAAGAACCATTACAACTACGGCTGGTTCTACTTCGGGGGCCTGTCGTTCATCCTGGCGGAAGTCATCGGAGTGCTTGCCGTGAATATCTACATCGAGAAGAACAAGGAGCTGCGGTTCAAGACGAAGCGGGAGTTTCTGAAGGCCTCCAGCAGCAGCCCATACGCCCGCATGCCGAGCTACAGGTACCGGCGGCGGCGCTCAAGATCATCCTCCCGCTCGACTGAAGCATCCCCGAGCCGGGACGTGTCGCCCATGGGACTGAAGATTACCGGAGCCATCCCTATGGGCGAACTGAGCATGTACACTCTGTCAAGAGAACCACTCAAAGTGACCACCGCCGCCTCCTACTCGCCGGATCAGGAAGCATCCTTCCTGCAAGTCCACGACTTCTTCCAACAAGACCTGAAGGAAGGATTCCACGTGTCTATGCTCAACCGAAGGACTACCCCCGTGTAGTGA

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYNYGWFYFGGLSFILAEVIGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV..

C13 (888888848)

ATGGAGTCACTGAAGAGATGGAACGAAGAAAGAGGATTGTGGTGTGAAAAGGGAGTCCAAGTCCTGCTCACTACCGTGGGGGCTTTCGCCGCCTTTGGGCTCATGACCATTGCCATCTCCACCGACTACTGGCTGTACACCCGGGCCCTGATCTGCAATACCACTAACCTCACCGCGGGGGGGGACGACGGAACCCCGCACCGCGGCGGCGGAGGCGCTAGCGAAAAGAAGGACCCAGGCGGCTTGACCCATTCGGGCCTGTGGAGGATCTGCTGTCTGGAGGGGCTGAAGCGGGGTGTCTGCGTGAAGATTAACCACTTCCCTGAGGACACTGACTACGATCACGACTCCGCCGAGTATCTTCTGCGCGTCGTGCGGGCCTCCTCGATTTTCCCCATCCTGTCCGCCATCCTGCTGCTCCTGGGTGGAGTGTGCGTGGCAGCGTCACGCGTGTACAAGTCCAAAAGGAACATCATCCTTGGTGCTGGAATCCTGTTTGTGGCCGCCGGGCTGTCCAACATTATTGGAGTGATTGTGTACATCAGCGCAAACGCCGGAGAGCCCGGCCCGAAGCGCGATGAAGAAAAGAAGAACCATTACAGCTACGGCTGGTCGTCCTTCTACTTCGGGGCGCTGTCGTTCATCGTGGCGGAAACCGTCGGAGTGCTTGCCGTGAATATCTACATCGAGAGGTCCAGAGAGGCACACTGCCAGTCCCGGAGCGACCTCCTGAAGGCCGGTGGAGGGGCCGGAGGCTCCGGAGGCAGCGGCCCATCGGCCATTCTGCGCCTGCCGAGCTACAGGTTTCGGTATCGGCGGCGCTCAAGATCATCCTCCCGCTCGTCCGAACCCTCCCCGAGCCGGGACGCCTCGCCCGGAGGACCGGGTGGCCCGGGATTCGCCTCCACCGACATCAGCATGTACACTCTGTCAAGAGATCCAAGCAAAGGAAGCGTCGCCGCGGGCCTCGCTGGCGCCGGAGGGGGCGGGGGTGGAGCAGTGGGAGCCTTCGGGGGCGCCGCTGGAGGAGCCGGGGGCGGTGGCGGAGGAGGCGGCGGAGCCGGTGCCGAGCGCGATCGGGGGGGCGCATCCGGTTTCCTGACGCTGCACAACGCGTTCCCGAAGGAAGCGGGCGGAGGAGTGACCGTGACTGTGACCGGACCGCCTGCCCCGCCTGCGCCTGCGCCCCCTGCTCCTTCCGCGCCTGCCCCCGGAACTCTCGCCAAGGAGGCCGCCGCCTCTAACACCAACACACTCAACCGAAAGACTACCCCCGTGTAGTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSSFYFGALSFIVAETVGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV..

C14 (488888888)

atggtgcgatgcgaccgcgggctgcagGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MVRCDRGLQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

C15 (448888888)

atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MVRCDRGLQMLLTTAGAFAAFSLMAIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV


11

C16 (444488888)

atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactggctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctgtggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtacctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagccgcaagaacaacATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

C17 (444448888)

atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactggctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctgtggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtacctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagccgcaagaacaacATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

C18 (444444888)

atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactggctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctgtggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtacctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagccgcaagaacaacatcgtcctcagtgccggcatcctcttcgtggctgcaggcctcagtaacatcatcggtatcatcgtctacattAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGIIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

C19 (444444488)

AtggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactggctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctgtggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtacctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagccgcaagaacaacatcgtcctcagtgccggcatcctcttcgtggctgcaggcctcagtaacatcatcggtatcatcgtctacatttccagcaacacaggtgacccgagtgacaagcgggacgaagacaaaaagaaccattacaactacggctggtctTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

C20 (444448484)

atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactggctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctgtggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtacctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagccgcaagaacaacATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTtccagcaacacaggtgacccgagtgacaagcgggacgaagacaaaaagaaccattacaactacggctggtctttttactttggaGGACTGTCTTTTATCCTGGCAGAAGTCATTggcgtcctggctgtaaacatttacattgagaaaaataaagagttgaggtttaagaccaaacgggaattccttaaggcgtcttcctcttctccttatgccaggatgccgagctacaggtaccggcgacggcgctcgaggtccagctcaaggtccaccgaggcctcgccctccagggacgtgtcgcccatgggcctgaagatcacaggggccatccccatgggggagctgtccatgtacacgctgtccagggagcccctcaaggtgaccaccgcagccagctacagccccgaccaggaggccagcttcctgcaggtgcatgactttttccagcaggacctgaaggaaggtttccacgtcagcatgctgaaccgacggacgacccctgtgtga

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIILGAGILFVAAGLSNIIGVIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGGLSFILAEVIGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV

C21 (444448444)

atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactggctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctgtggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtacctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagccgcaagaacaacATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTtccagcaacacaggtgacccgagtgacaagcgggacgaagacaaaaagaaccattacaactacggctggtctttttactttggagctctgtctttcattgtggctgagaccgtgggcgtcctggctgtaaacatttacattgagaaaaataaagagttgaggtttaagaccaaacgggaattccttaaggcgtcttcctcttctccttatgccaggatgccgagctacaggtaccggcgacggcgctcgaggtccagctcaaggtccaccgaggcctcgccctccagggacgtgtcgcccatgggcctgaagatcacaggggccatccccatgggggagctgtccatgtacacgctgtccagggagcccctcaaggtgaccaccgcagccagctacagccccgaccaggaggccagcttcctgcaggtgcatgactttttccagcaggacctgaaggaaggtttccacgtcagcatgctgaaccgacggacgacccctgtgtga

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIILGAGILFVAAGLSNIIGVIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGALSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV

D1 (282828282)

ATGGGTCTGTTCGACAGAGGTGTGCAGGTGCTGCTGACTACTGTTGGAGCTTTTGCAGCTTTTGGACTGATGACCATTGCTATCAGCACTGACTACTGGCTGTACAGCAGAGGAGTGTGCAAGACCAAGAGTGTGAGTGAGAACGAGACCAGCAAGAAGAACGAGGAGGTGATGACTCACAGTGGTCTGTGGAGAACCTGTTGTCTGGAAGGTAACTTCAAAGGTCTGTGCAAGCAGATCGACCACTTTCCAGAGGACGCTGACTACGAGGCTGACACTGCAGAGTACTTCCTGAGAGCTGTGAGAGCTAGTAGCATCTTTCCAATCCTGAGTGCTATCCTGCTGCTGCTTGGAGGTGTGTGTGTTGCAGCTAGCAGAGTGTACAAGACCAGACACAACATCATCCTTGGAGCTGGAATCCTGTTCGTTGCAGCTGGACTTAGCAACATCATTGGAGTGATCGTGTACATCAGTGCTAATGCAGGTGATCCAAGCAAGAGTGACAGCAAGAAGAACAGCTACAGCTATGGTTGGAGCTTCTACTTTGGAGGTCTGAGCTTCATCCTTGCTGAGGTGATTGGTGTTCTTGCTGTGAACATCTACATCGAGAGACACAAGCAGCTGAGAGCTACTGCTAGAGCTACTGACTACCTGCAAGCTAGTGCTATCACCAGAATCCCTAGCTACAGATACAGATACCAGAGAAGAAGCAGAAGCAGTAGCAGAAGTACTGAACCTAGTCACAGCAGAGATGCCAGTCCTGTAGGTATCAAAGGTTTCAACACTCTTCCAAGCACTGAGATCAGCATGTACACTCTGAGCAGAGATCCTCTGAAAGCTGCAACCACTCCTACTGCAACCTACAACAGCGACAGAGACAACAGCTTCCTGCAGGTGCACAACTGCATCCAGAAGGAGAACAAGGACAGTCTGCACAGCAACACTGCTAACAGAAGAACCACTCCAGTGTGA

MGLFDRGVQVLLTTVGAFAAFGLMTIAISTDYWLYSRGVCKTKSVSENETSKKNEEVMTHSGLWRTCCLEGNFKGLCKQIDHFPEDADYEADTAEYFLRAVRASSIFPILSAILLLLGGVCVAASRVYKTRHNIILGAGILFVAAGLSNIIGVIVYISANAGDPSKSDSKKNSYSYGWSFYFGGLSFILAEVIGVLAVNIYIERHKQLRATARATDYLQASAITRIPSYRYRYQRRSRSSSRSTEPSHSRDASPVGIKGFNTLPSTEISMYTLSRDPLKAATTPTATYNSDRDNSFLQVHNCIQKENKDSLHSNTANRRTTPV


12

Supplemental Table 7. The primers used for generation of the point mutations of CACNG8 and CACNG4.

5’ end 3’ end Full length mutations Forward

primer Reverse primer


Reverse primer


Reverse primer

Template

CACNG8-G210A

P61 P62 CACNG8 P63 P64 CACNG8 P61 P64 5’ end + 3’ end

CACNG8-L215V


CACNG8-V218T


CACNG8-I219V


CACNG8-I159V


CACNG8-G161S


CACNG8-V177I


CACNG8-N173A


CACNG8-I174A


CACNG8-I175A


CACNG8-G176A


CACNG8-G209A


CACNG8-L211A


CACNG8-S212A


CACNG8-F213A


CACNG8-I214A


CACNG8-V177I, G210A

P61 P62 CACNG8-V177I

P63 P64 CACNG8-V177I

P61 P64 5’ end + 3’ end

CACNG4-A189G


CACNG4-I156V, A189A

P97 P101 CACNG4-A189G

P102 P100 CACNG4-A189G

P97 P100 5’ end + 3’ end

Primer sequences (5’ TO 3’) P61 ACGTCAGAATTCGCCACCATGGAGAGCCTGAAGCGGTGGAACGA P62 ACTTCTGCCAGGATAAAAGACAGAGCCCCAAAGTAGAAAGACCA P63 TGGTCTTTCTACTTTGGGGCTCTGTCTTTTATCCTGGCAGAAGT


13

P64 ACGTCAGCGGCCGCTCAGACGGGTGTGGTTTTTCGGTTCAGTGTATTGG P65 CAGCACCCCAATGACTTCTGCTACGATAAAAGACAGTCC P66 GGACTGTCTTTTATCGTAGCAGAAGTCATTGGGGTGCTG P67 ATTGACGGCCAGCACCCCAATAGTTTCTGCCAGGATAAAAGAC P68 GTCTTTTATCCTGGCAGAAACTATTGGGGTGCTGGCCGTCAAT P69 ATAGATATTGACGGCCAGCACCCCGACGACTTCTGCCAGGATAAAAGA P70 TCTTTTATCCTGGCAGAAGTCGTCGGGGTGCTGGCCGTCAATATCTAT P71 GAACAGAATGCCGGCTCCCAGAACGATATTTCTTTTGGACTTGTA P72 TACAAGTCCAAAAGAAATATCGTTCTGGGAGCCGGCATTCTGTTC P73 CACGAACAGAATGCCGGCACTCAGAATGATATTTCTTTTGGA P74 TCCAAAAGAAATATCATTCTGAGTGCCGGCATTCTGTTCGTG P75 CATTGGCGCTAATATAGACGATAATGCCAATGATGTTTGACAGT P76 ACTGTCAAACATCATTGGCATTATCGTCTATATTAGCGCCAATG P77 AATATAGACGATCACGCCAATGATTGCTGACAGTCCTGCA P78 TGCAGGACTGTCAGCAATCATTGGCGTGATCGTCTATATT P79 ATAGACGATCACGCCAATTGCGTTTGACAGTCCTGCA P80 TGCAGGACTGTCAAACGCAATTGGCGTGATCGTCTAT P81 CTAATATAGACGATCACGCCTGCGATGTTTGACAGTCCT P82 AGGACTGTCAAACATCGCAGGCGTGATCGTCTATATTAG P83 CTAATATAGACGATCACTGCAATGATGTTTGACAGTC P84 GACTGTCAAACATCATTGCAGTGATCGTCTATATTAG P85 TTGGCGCTAATATAGACTGCCACGCCAATGATGTTTGAC P86 GTCAAACATCATTGGCGTGGCAGTCTATATTAGCGCCAA P87 CAGGATAAAAGACAGTCCTGCAAAGTAGAAAGACCA P88 TGGTCTTTCTACTTTGCAGGACTGTCTTTTATCCTG P89 CTTCTGCCAGGATAAAAGATGCTCCCCCAAAGTAGAAAG P90 CTTTCTACTTTGGGGGAGCATCTTTTATCCTGGCAGAAG P91 GACTTCTGCCAGGATAAATGCCAGTCCCCCAAAGTAG P92 CTACTTTGGGGGACTGGCATTTATCCTGGCAGAAGTC P93 CAATGACTTCTGCCAGGATTGCAGACAGTCCCCCAAAG P94 CTTTGGGGGACTGTCTGCAATCCTGGCAGAAGTCATTG P95 CAATGACTTCTGCCAGTGCAAAAGACAGTCCCCCAAA P96 TTTGGGGGACTGTCTTTTGCACTGGCAGAAGTCATTG P97 ACGTCAGAATTCGCCACCATGGTGCGATGCGACCGCGGGCTGCAGA P98 CAGCCACAATGAAAGACAGTCCTCCAAAGTAAAAAGACCAG P99 CTGGTCTTTTTACTTTGGAGGACTGTCTTTCATTGTGGCTG P100 ATCTGTGCGGCCGCTCACACAGGGGTCGTCCGTCGGTTCAGCAT P101 GGAAATGTAGACGATGACACCGATGATGTTACTGAG P102 CTCAGTAACATCATCGGTGTCATCGTCTACATTTCC


14

Supplemental Table 8. DNA and protein sequences for CACNG8 and CACNG4 point mutants.

construct DNA Sequence protein sequence CACNG8-G210A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGgctCTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGALSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-L215V

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCgtaGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFIVAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-V218T

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAactATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAETIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-I219V

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCgtcGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVVGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-I159V

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCgttCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIVLGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-G161S

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGagtGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILSAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-V177I

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCattATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGIIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV


15

CACNG8-N173A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAgcaATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSAIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-I174A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACgcaATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNAIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-I175A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCgcaGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIAGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-G176A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTgcaGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIAVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-G209A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTgcaGGACTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFAGLSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-L211A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGAgcaTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGASFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-S212A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGgcaTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLAFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV


16

CACNG8-F213A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTgcaATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSAILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-I214A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCGTGATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGGGACTGTCTTTTgcaCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGVIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGGLSFALAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG8-V177I, G210A

ATGGAGAGCCTGAAGCGGTGGAACGAGGAACGAGGGCTGTGGTGCGAAAAAGGAGTGCAGGTCCTGCTGACCACAGTGGGAGCCTTCGCCGCTTTTGGGCTGATGACAATCGCTATTTCCACTGACTACTGGCTGTATACCCGAGCACTGATCTGTAACACTACCAATCTGACTGCCGGCGGGGACGATGGAACCCCTCACAGAGGAGGAGGAGGAGCATCCGAGAAGAAAGATCCAGGCGGGCTGACACATTCTGGCCTGTGGAGGATCTGCTGTCTGGAAGGACTGAAGCGCGGCGTGTGCGTCAAAATTAACCACTTCCCCGAGGACACTGATTACGACCATGATAGTGCCGAATATCTGCTGCGAGTGGTCCGGGCTAGCTCCATCTTTCCTATTCTGTCAGCAATCCTGCTGCTGCTGGGAGGCGTGTGCGTGGCAGCAAGCCGGGTGTACAAGTCCAAAAGAAATATCATTCTGGGAGCCGGCATTCTGTTCGTGGCTGCAGGACTGTCAAACATCATTGGCattATCGTCTATATTAGCGCCAATGCAGGGGAGCCCGGACCTAAGAGAGACGAGGAAAAGAAAAACCACTACTCCTATGGCTGGTCTTTCTACTTTGGGgcaCTGTCTTTTATCCTGGCAGAAGTCATTGGGGTGCTGGCCGTCAATATCTATATTGAGCGCAGTCGAGAAGCCCATTGCCAGAGCCGGAGCGACCTGCTGAAAGCTGGAGGAGGAGCAGGAGGATCCGGAGGATCTGGCCCAAGTGCCATCCTGAGGCTGCCCTCTTACCGGTTCAGATATCGGAGAAGGAGTAGATCTAGTTCAAGGAGCTCCGAGCCATCACCCAGCCGCGATGCTTCTCCAGGAGGACCTGGAGGACCAGGATTTGCCAGTACCGACATCTCAATGTACACACTGTCCCGGGATCCATCAAAGGGCAGCGTGGCCGCTGGCCTGGCAGGAGCTGGAGGAGGAGGAGGAGGAGCAGTGGGCGCCTTCGGAGGCGCAGCCGGGGGAGCTGGCGGGGGAGGAGGCGGAGGCGGGGGAGCAGGGGCCGAGAGGGATCGCGGCGGGGCCAGCGGATTCCTGACCCTGCACAATGCCTTTCCCAAGGAAGCTGGAGGAGGAGTGACAGTCACTGTGACCGGACCACCTGCTCCACCAGCTCCAGCACCTCCAGCACCTTCCGCCCCTGCTCCAGGCACACTGGCCAAGGAGGCAGCAGCATCAAATACCAATACACTGAACCGAAAAACCACACCCGTCTGA

MESLKRWNEERGLWCEKGVQVLLTTVGAFAAFGLMTIAISTDYWLYTRALICNTTNLTAGGDDGTPHRGGGGASEKKDPGGLTHSGLWRICCLEGLKRGVCVKINHFPEDTDYDHDSAEYLLRVVRASSIFPILSAILLLLGGVCVAASRVYKSKRNIILGAGILFVAAGLSNIIGIIVYISANAGEPGPKRDEEKKNHYSYGWSFYFGALSFILAEVIGVLAVNIYIERSREAHCQSRSDLLKAGGGAGGSGGSGPSAILRLPSYRFRYRRRSRSSSRSSEPSPSRDASPGGPGGPGFASTDISMYTLSRDPSKGSVAAGLAGAGGGGGGAVGAFGGAAGGAGGGGGGGGGAGAERDRGGASGFLTLHNAFPKEAGGGVTVTVTGPPAPPAPAPPAPSAPAPGTLAKEAAASNTNTLNRKTTPV

CACNG4-A189G

atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactggctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctgtggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtacctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagccgcaagaacaacatcgtcctcagtgccggcatcctcttcgtggctgcaggcctcagtaacatcatcggtatcatcgtctacatttccagcaacacaggtgacccgagtgacaagcgggacgaagacaaaaagaaccattacaactacggctggtctttttactttggaGGActgtctttcattgtggctgagaccgtgggcgtcctggctgtaaacatttacattgagaaaaataaagagttgaggtttaagaccaaacgggaattccttaaggcgtcttcctcttctccttatgccaggatgccgagctacaggtaccggcgacggcgctcgaggtccagctcaaggtccaccgaggcctcgccctccagggacgtgtcgcccatgggcctgaagatcacaggggccatccccatgggggagctgtccatgtacacgctgtccagggagcccctcaaggtgaccaccgcagccagctacagccccgaccaggaggccagcttcctgcaggtgcatgactttttccagcaggacctgaaggaaggtttccacgtcagcatgctgaaccgacggacgacccctgtgtga

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGIIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGGLSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV

CACNG4-I156V, A189A

atggtgcgatgcgaccgcgggctgcagatgctgctgaccacggccggagccttcgccgccttctcgctcatggccatcgccatcggcaccgactactggctgtactccagcgcgcacatctgcaacggcaccaacctgaccatggacgacgggcccccgccccgccgcgcccgcggcgacctcacccactctggtctgtggcgggtgtgctgcatcgaagggatctataaagggcactgcttccggatcaatcacttcccagaggacaatgactacgaccacgacagctcggagtacctcctccgcatcgtgcgagcctccagcgtcttccccatcctcagcaccatcctgctcctgctgggtggcctgtgcatcggtgctggcaggatctacagccgcaagaacaacatcgtcctcagtgccggcatcctcttcgtggctgcaggcctcagtaacatcatcggtGTCatcgtctacatttccagcaacacaggtgacccgagtgacaagcgggacgaagacaaaaagaaccattacaactacggctggtctttttactttggaGGActgtctttcattgtggctgagaccgtgggcgtcctggctgtaaacatttacattgagaaaaataaagagttgaggtttaagaccaaacgggaattccttaaggcgtcttcctcttctccttatgccaggatgccgagctacaggtaccggcgacggcgctcgaggtccagctcaaggtccaccgaggcctcgccctccagggacgtgtcgcccatgggcctgaagatcacaggggccatccccatgggggagctgtccatgtacacgctgtccagggagcccctcaaggtgaccaccgcagccagctacagccccgaccaggaggccagcttcctgcaggtgcatgactttttccagcaggacctgaaggaaggtttccacgtcagcatgctgaaccgacggacgacccctgtgtga

MVRCDRGLQMLLTTAGAFAAFSLMAIAIGTDYWLYSSAHICNGTNLTMDDGPPPRRARGDLTHSGLWRVCCIEGIYKGHCFRINHFPEDNDYDHDSSEYLLRIVRASSVFPILSTILLLLGGLCIGAGRIYSRKNNIVLSAGILFVAAGLSNIIGVIVYISSNTGDPSDKRDEDKKNHYNYGWSFYFGGLSFIVAETVGVLAVNIYIEKNKELRFKTKREFLKASSSSPYARMPSYRYRRRRSRSSSRSTEASPSRDVSPMGLKITGAIPMGELSMYTLSREPLKVTTAASYSPDQEASFLQVHDFFQQDLKEGFHVSMLNRRTTPV


17

Supplemental Table 9. Selectivity of JNJ-55511118 and JNJ-56022486 against a panel of receptors using radioligand binding assays. Inhibition of binding by less than 10% is indicated by a dash. Data were generated by Cerep SA (http://www.cerep.fr/).

inhibition of radioligand binding (% control)

Assay Reference Compound 56022486 (1 µM)

55511118 (1µM)

A1 (h) (antagonist radioligand) DPCPX 12 39

A2A (h) (agonist radioligand) NECA 18 ‐

A3 (h) (agonist radioligand) IB‐MECA ‐ 11

alpha 1 (non‐selective) (antagonist radioligand) prazosin ‐ ‐

alpha 2 (non‐selective) (antagonist radioligand) yohimbine ‐ ‐

beta 1 (h) (agonist radioligand) atenolol ‐ ‐

AT1 (h) (antagonist radioligand) saralasin ‐ ‐

BZD (central) (agonist radioligand) diazepam ‐ ‐

B2 (h) (agonist radioligand) NPC 567 ‐ ‐

CCK1 (CCKA) (h) (agonist radioligand) CCK‐8s ‐ ‐

D1 (h) (antagonist radioligand) SCH 23390 12 ‐

D2S (h) (antagonist radioligand) (+)butaclamol ‐ ‐

ETA (h) (agonist radioligand) endothelin‐1 ‐ ‐

GABA (non‐selective) (agonist radioligand) GABA ‐ ‐

GAL2 (h) (agonist radioligand) galanin ‐ ‐

CXCR2 (IL‐8B) (h) (agonist radioligand) IL‐8 ‐ ‐

CCR1 (h) (agonist radioligand) MIP‐1a ‐ ‐

H1 (h) (antagonist radioligand) pyrilamine ‐ ‐

H2 (h) (antagonist radioligand) cimetidine ‐ ‐

MC4 (h) (agonist radioligand) NDP‐a‐MSH ‐ ‐

MT1 (ML1A) (h) (agonist radioligand) melatonin 57 19

M1 (h) (antagonist radioligand) pirenzepine ‐ ‐

M2 (h) (antagonist radioligand) methoctramine ‐ ‐

M3 (h) (antagonist radioligand) 4‐DAMP ‐ ‐

NK2 (h) (agonist radioligand) [Nleu10]‐NKA (4‐10) ‐ ‐

NK3 (h) (antagonist radioligand) SB 222200 ‐ ‐

Y1 (h) (agonist radioligand) NPY ‐ ‐

Y2 (h) (agonist radioligand) NPY ‐ ‐

NTS1 (NT1) (h) (agonist radioligand) neurotensin ‐ ‐

delta 2 (DOP) (h) (agonist radioligand) DPDPE ‐ ‐

kappa (KOP) (agonist radioligand) U 50488 ‐ ‐

mu (MOP) (h) (agonist radioligand) DAMGO ‐ ‐

NOP (ORL1) (h) (agonist radioligand) nociceptin ‐ ‐

5‐HT1A (h) (agonist radioligand) 8‐OH‐DPAT ‐ ‐

5‐HT1B (antagonist radioligand) serotonin ‐ ‐

5‐HT2A (h) (antagonist radioligand) ketanserin ‐ 20

5‐HT2B (h) (agonist radioligand) (±)DOI ‐ 78

5‐HT3 (h) (antagonist radioligand) MDL 72222 ‐ ‐

5‐HT5a (h) (agonist radioligand) serotonin ‐ 13

5‐HT6 (h) (agonist radioligand) serotonin ‐ ‐

5‐HT7 (h) (agonist radioligand) serotonin ‐ ‐

sst (non‐selective) (agonist radioligand) somatostatin‐14 ‐ ‐

VPAC1 (VIP1) (h) (agonist radioligand) VIP ‐ ‐

V1a (h) (agonist radioligand) [d(CH2)51,Tyr(Me)2]‐AVP ‐ ‐

Ca2+ channel (L, verapamil site) (antagonist radioligand) D 600 ‐ ‐

KV channel (antagonist radioligand) a‐dendrotoxin ‐ ‐

SKCa channel (antagonist radioligand) apamin ‐ ‐

Na+ channel (site 2) (antagonist radioligand) veratridine ‐ ‐

Cl‐ channel (GABA‐gated) (antagonist radioligand) picrotoxinin ‐ ‐

norepinephrine transporter (h) (antagonist radioligand) protriptyline ‐ ‐

dopamine transporter (h) (antagonist radioligand) BTCP ‐ ‐

5‐HT transporter (h) (antagonist radioligand) imipramine ‐ 10


18

Supplemental Table 10. Summary of pharmacokinetic parameters. ‘n.d.’ indicates that this parameter was not determined.

parameter JNJ‐55511118 JNJ‐56022486

rat mouse rat mouse

free fraction (brain) 0.24% n.d. 2.07% n.d.

free fraction (plasma) 1.48% 0.88% 7.59% 7.06%

oral bioavailability 133% n.d. 93% n.d.

clearance (mL/min/kg) 4.8 n.d. 11.4 n.d.

Vss (L/kg) 1.8 n.d. 1.8 n.d.

t1/2 (min) 260 n.d. 109 n.d.

BBB 2.2 1.8 0.41 n.d.

Kpuu 0.35 0.49 0.11 n.d.

Tmax (10mg/kg, p.o.) (min) 120 60 120 n.d.

Cmax (10mg/kg, p.o.) (ng/mL) 1678 3678 1819 n.d.

maximum occupancy (%) 73% 83% 41% n.d.


19

Supplemental Figures

Supplemental Figure 1. Synthesis of JNJ-55511118. To a solution of (2-chloro-6-(trifluoromethoxy)phenyl)boronic acid (4.5g, 19 mmol) and 5-bromo-1H-benzo[d]imidazol-2(3H)-one (2.0 g, 9.4 mmol) in 4:1 dioxane:water (20 ml) were added potassium phosphate (4.0 g, 19 mmol) and 1,1’-bis(di-tert-butylphosphino)ferrocene palladium (II) chloride (612 mg, 0.94 mmol). The mixture was degassed with nitrogen and then heated at 100 °C for 16h. After cooling to room temperature, the reaction mixture was filtered through celite and washed successively with EtOAc and DCM. The filtrate was combined with silica gel, concentrated in vacuo, and dry-loaded onto an 80g silica gel cartridge. Purification by flash chromatography (0-10% MeOH in DCM) afforded a solid. The solid was dissolved in hot EtOH (60 °C), and water was added to initiate precipitation. After cooling to room temperature, the precipitate was filtered and dried under high vacuum for 4h to provide the desired product as a white solid (918 mg, 30% yield). 1H NMR (400 MHz, DMSO-d6): δ 10.75 (s, 1H), 10.69 (s, 1H), 7.69 – 7.60 (m, 1H), 7.59 – 7.43 (m, 2H), 7.08 – 6.98 (m, 1H), 6.86 – 6.77 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 155.3, 146.9, 134.52, 134.47, 129.9, 129.7, 129.5, 128.6, 125.1, 122.1, 119.9, 119.8 (q, J = 257 Hz), 109.6, 108.2. Anal. Calc’d. for C14H18ClF3N2O2: C, 51.16; H, 2.45; N, 8.52. Found: C, 51.34; H, 2.07; N, 8.41.


20

NH

HN

OCl

I

Cl

c

aNH

HN

O

B

O

O

a) NBS, ACBN, CCl4 90oC

b) KCN, DMF/H2O 40oC

c) cat PdCl2(dppf)-CH2Cl2, K3PO4

dioxane/H2O 100 oC

d) NBS, TFA

e) 3H-H3, Pd/C, MeOH

CN

I

Cl

Br

I

Cl

CNb

NH

HN

OCl

CN

Br

d

NH

HN

OCl

CN

T

e

Supplemental Figure 2. Synthesis and tritiation of JNJ-56022486. (a) 1-(bromomethyl)-3-chloro-2-iodobenzene. To a solution of 1-chloro-2-iodo-3-methylbenzene (4.0 g, 16 mmol) in CCl4 (12 mL), were added N-bromosuccinimide (5.6 g, 32 mmol) and 1,1'-azobis(cyclohexanecarbonitrile) (3.9 g, 16 mmol). The mixture was degassed with nitrogen and then heated at 90 °C for 1h. After cooling to room temperature, silica gel was added, and the solvent was removed in vacuo. Purification by flash column chromatography (SiO2; 0 - 5% EtOAc /hexanes) provided the title compound as an oil (3.7 g, 70% yield). 1H NMR (400 MHz, CDCl3): δ 7.37 (m, 2H), 7.29 – 7.23 (m, 1H), 4.65 (s, 2H). (b) 2-(3-chloro-2-iodophenyl)acetonitrile. To a solution of 1-(bromomethyl)-3-chloro-2-iodobenzene (1.0 g, 3.0 mmol) in DMF (13 mL) was added a solution of potassium cyanide (236 mg, 3.6 mmol) in water (1.3 mL). After stirring at 40 °C for 1h, the reaction mixture was cooled to room temperature, diluted with water, and extracted with EtOAc (2x). The combined organic extracts were dried over Na2SO4, and concentrated to obtain the product as a white solid (770 mg, 92% yield). 1H NMR (500 MHz, CDCl3): δ 7.44 (m, 2H), 7.36 – 7.33 (m, 1H), 3.93 – 3.83 (m, 2H). (c) 2-[3-chloro-2-(2-oxo-1,3-dihydrobenzimidazol-5-yl)phenyl]acetonitrile. To a solution of 2-(3-chloro-2-iodophenyl)acetonitrile (448 mg, 1.6 mmol) and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazol-2(3H)-one (350 mg, 1.4 mmol) in 4:1 dioxane:water (3.5 ml) were added potassium phosphate (571 mg, 2.7 mmol) and [1,1’-bis(diphenylphosphino)ferrocene]dichloropalladium (II) dichloromethane complex (98 mg, 0.13 mmol). The mixture was degassed with nitrogen and then heated at 100 °C for 16h. After cooling to room temperature, the reaction mixture was diluted with water and extracted with DCM (x3). The combined organic extracts were dried over Na2SO4 and filtered. Silica gel was added to the filtrate, and the solvent was removed in vacuo. The resulting solid was dry-loaded onto a 40g silica gel cartridge and purified by flash chromatography (0-50% 2M NH3 in MeOH/ DCM) to afford the desired product as a solid (211 mg, 55% yield). MS (ESI): mass calcd. for C15H10ClN3O, 283.1; m/z found, 284.1 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.74 (s, 1H), 10.72 (s, 1H), 7.56 (dd, J = 7.9, 1.4 Hz, 1H), 7.53 – 7.49 (m, 1H), 7.48 –


21

7.40 (m, 1H), 7.06 – 7.00 (m, 1H), 6.78 – 6.72 (m, 2H), 3.69 (s, 2H). 13C NMR (150 MHz, DMSO-d6): δ 155.3, 140.2, 133.8, 132.3, 129.9, 129.5, 129.3, 128.9, 128.4, 127.6, 121.3, 118.6, 109.1, 108.6, 22.0. (d) 2-[2-(6-bromo-2-oxo-1,3-dihydrobenzimidazol-5-yl)-3-chloro-phenyl]acetonitrile. To a solution of 2-[3-chloro-2-(2-oxo-1,3-dihydrobenzimidazol-5-yl)phenyl]acetonitrile (210 mg, 0.74 mmol) in TFA (7.4 mL) was added N-bromosuccinimide (132 mg, 0.74 mmol). After stirring at room temperature for 24h, the solvent was removed in vacuo. Residual TFA was removed by trituration with DCM and concentration under reduced pressure. The resulting solid was diluted with saturated aqueous NaHCO3 and extracted twice with EtOAc. The combined organic extracts were dried over Na2SO4 and concentrated in vacuo. Purification by reverse-phase HPLC (0.05M NH4OH in water/MeCN) afforded the desired product as a white solid (107 mg, 40% yield). MS (ESI): mass calcd. for C15H9BrClN3O, 361.0; m/z found, 362.0 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.92 (bs, 2H), 7.63 – 7.42 (m, 3H), 7.32 – 7.21 (s, 1H), 6.85 – 6.74 (s, 1H), 3.67 – 3.57 (m, 2H). (e) 2-[3-chloro-2-(2-oxo-6-tritio-1,3-dihydrobenzimidazol-5-yl)phenyl]acetonitrile. This step was performed by Moravek Biochemicals, Inc. (Brea, CA). A 2 cc round-bottom flask was charged with 2-[2-(6-bromo-2-oxo-1,3-dihydrobenzimidazol-5-yl)-3-chloro-phenyl]acetonitrile (5 mg), 10 wt% Pd/C (5 mg), MeOH (0.5 ml), and tritium gas (10 Ci). The mixture was stirred for 6h at room temperature. The crude product was dissolved in EtOH and filtered. The labile tritium was exchanged as the EtOH was removed in vacuo. This was repeated 2 additional times. The crude product was purified by reverse-phase HPLC (Gemini 5 μm C-18 column, 35% aq acetonitrile containing 0.01% NH4OH) to afford the title compound. The specific activity was determined to be 22.3 Ci/mmol, and the product was stored at -20 °C in EtOH at a concentration of 1.0 mCi/mL. MS (FTMS + c NSI SIM): mass calcd. for C15H9TClN3O, 285.1; m/z found, 286.1 [M+H]+.


22

Supplemental Figure 3. Schematic drawings of the chimeric proteins. Chimeras are labelled by their shorthand notation, with the ‘C’ prefix designating a chimera of TARP-8 and -4 and the ‘D’ prefix designating a chimera of TARP-8 and -2. Also shown is the nine-digit notation; each digit represents the TARP sequence for that section of the protein with the sequence NT, TM1, EX1, TM2, IN1, TM3, EX2, TM4, CT. The actual splice points used are listed in Supplemental Table 4. The drawing represent the predicted topology of the protein, with the colors representing the TARP sequence used for that segment: TARP-8 (blue), TARP-4 (red), TARP-2 (green).

444444444

C2444444448

8888888888

C1888888884

C3888888844

C4888888444

C5888884444

C6888844444

C7888444444

C8884444444

C9844444444

C14488888888

C15448888888

C16444888888

C17444488888

C18444448888

C19444444888

C11448444444

C10884888888

C21444448444

C20444448484

C13888888848

C12444444484

D1282828282

2222222222


23

Supplemental Figure 4. Inhibition of peak currents by 1 µM JNJ-55511118 in cells expressing AMPA receptors (N = 4-8 per group). The box plots indicate the median value (line), the mean (square), the 25th–75th percentiles (box) and the minimum-maximum (whiskers).

Glu

A1

o -

8

Glu

A1

i +

8

Glu

A1

o -

2

Glu

A1

i +

2

Glu

A1o

hip

po

(+

/+)

cere

bellu

m

hip

po

(-/

-)

20

40

60

80

100

120

peak

cur

rent

(%

)


24

Supplemental Figure 5. Activity of JNJ-55511118 in hippocampal slices in whole-cell mode. (A) Representative eEPSC recordings prior to (black) and after (red) bath application of 1 µM JNJ-55511118. These traces are the average of five consecutive sweeps. Holding potential was -70 mV to isolate the AMPAR currents. (B) AMPAR-mediated eEPSCs in response to 50 Hz burst stimulation show reduced summation in the presence of 1 µM JNJ-55511118. (C) NMDAR-mediated currents were isolated by holding the cell ay +40mV; these currents were unaffected by 1 µM JNJ-55511118. (D) Paired-pulse ratio (ratio of the response to a pulse, divided by the response to the immediately preceding pulse) calculated from the data in (B). The paired-pulse ratio was unaffected by 1 µM JNJ-55511118, indicating a post-synaptic site of action for the drug.

P2/P1 P3/P2 P4/P3 P5/P4

0.5

1.0

1.5

2.0

2.5

3.0

paire

d-pu

lse

ratio

Pulse pairs

control JNJ-55511118

control JNJ-55511118 (1uM)

100 pA

20 ms

control 55511118

50 pA

200 ms

control JNJ-55511118 (1 µM)

50 pA

20 ms

BA

DC


25

Supplemental Figure 6. Activity of JNJ-55511118 in GluA1i + TARP-8 with and without cyclothiazide (CTZ; 100 µM). (A) Time courses of the current in outside-out patches from HEK cells transiently transfected with GluA1i + TARP-8, in the continuous presence of cyclothiazide to remove desensitization. Solid lines are the average of five recordings prior to (black) and after (blue) exposure to 1 µM JNJ-55511118. (B) Average glutamate-evoked peak (black) and steady-state (red) currents in six patches, normalized to the corresponding current prior to the addition of 1 µM JNJ-55511118. Also shown are the steady-state currents from patches exposed to cyclothiazide (blue). The box plots show the median (line), mean (square), 25th and 75th percentiles (box), minimum/maximum (whiskers), and individual data (solid circles).

peak steady-state plus CTZ0.0

0.2

0.4

0.6

0.8

1.0

I (J

NJ-

5551

1118

/con

trol

)

control JNJ-55511118

CTZGlu

100 pA

100 ms

B

A


26

Supplemental Figure 7. Pharmacokinetics and target occupancy for JNJ-56022486 in rat. (A) Plasma concentration as a function of time in Sprague-Dawley rats following oral (5 mg/kg p.o.) or intravenous (1 mg/kg i.v.) administration. (B) Brain and plasma concentrations in rat after a 10 mg/kg p.o. dose. Target occupancy was determined by autoradiography (ARG) of brain slices.

0 6 12 18 2410

100

1000

10000

plasma brain occupancy

conce

ntra

tion (

ng/

mL)

time (hr)

0

20

40

60

80

100

rat

occ

upa

ncy

(perc

ent)

0 6 12 18 24

1

10

100

1000

p.o. (5 mg/kg) i.v. (1 mg/kg)

conce

ntra

tion (

ng/

mL)

time (hr)

ratA B


27

A

B

C

D E

Supplemental Figure 8. Dose response effects of JNJ-55511118 (1, 3, and 10 mg/kg, p.o.) on sleep parameters in rats. (A) Wake duration, (B) NREM duration, (C) REM duration, (D) NREM latency, and (E) REM latency were determined for the 8-hour period after compound or vehicle administration. Data are expressed in minutes and are represented as means ± SEM of the same 8 animals per dose. Oral administration of JNJ-55511118 in rats induced a dose-dependent increase in wake duration [F(9, 54) = 2.79,


28

p = 0.009] and a decrease in the duration of both NREM [F(9, 54) = 2.27, p = 0.031] and REM [F(9, 54) = 1.95, p = 0.064] sleep. In addition, a dose-dependent increase in NREM latency [F(3, 18) = 9.61, p < 0.001] and in REM latency [F(3, 18) = 5.67, p = 0.007] was observed. At 10 mg/kg, JNJ-55511118 produced a significant wake-promoting effect for 4 hours and reduced the time spent in NREM sleep for 2 hours and in REM sleep for 6 hours following the treatment, associated with a significant delay to the onset of both NREM and REM sleep. * p < 0.05 ** p < 0.01 and *** p < 0.001 versus vehicle, based on one-way or two-way ANOVA followed by Dunnett’s multiple comparison test.


29

A B

C D

E F

G H

Supplemental Figure 9. EEG effects JNJ-55511118 in sleep states. (A-D) Dose response effects of

JNJ-55511118 (1, 3, and 10 mg/kg, p.o.) on EEG power activity during NREM sleep in rats. JNJ-55511118 produced a dose-dependent decrease in EEG power in the entire 1-30 Hz frequency range during NREM sleep. Delta (1-4 Hz), theta (4-10 Hz), sigma (10-15 Hz) and beta (15-30 Hz) EEG oscillations were significantly reduced from the dose of 1 mg/kg onwards. (E-H) Dose response effects of JNJ-55511118 (1, 3, and 10 mg/kg, p.o.) on EEG power activity during REM sleep in rats. During REM sleep, delta (1-4 Hz), theta (4-10 Hz) and beta (15-30 Hz) EEG oscillations were significantly reduced from the dose of 1 mg/kg onwards. In contrast, EEG oscillations in the sigma frequency range (10-15 Hz) were minimally affected, except for a small increase at the dose of 10 mg/kg. * p < 0.05 ** p < 0.01 and *** p < 0.001 versus vehicle, based on one-way or two-way ANOVA followed by Dunnett’s multiple comparison test.


30

vehicle 10mg/kg0

50

100

150

200af

terd

isch

arg

e d

urat

ion

(s)

Supplemental Figure 10. Effects of JNJ-55511118 on amygdala kindling in rat. The box plot shows the median (line), mean (square), 25th and 75th percentiles (box), minimum/maximum (whiskers), and individual data (solid circles).


31

0

200

400

600

800

1000

1200

1400

1600

1800

1 2 3 1 2 3 1 2 3 1 2 3 D1 D2 D3 D4

D1 D2 D3 D4 Average per Day

Distance (cm

)Pathlength

0mg/kg 0.63mg/kg 2.5mg/kg 10mg/kg

****

***

**

*

0

10

20

30

40

50

60

70

80

90

100

1 2 3 1 2 3 1 2 3 1 2 3 D1 D2 D3 D4


Time %

% time in periphery

0mg/kg 0.63mg/kg 2.5mg/kg 10mg/kg

**

0

5

10

15

20

25

30

35

1 2 3 1 2 3 1 2 3 1 2 3 D1 D2 D3 D4


Velocity (cm

/s)

Swim speed0mg/kg 0.63mg/kg 2.5mg/kg 10mg/kg

**

***

* ***

A

B

C

Supplemental Figure 11. Additional measures in the Morris Water Maze test. In each case, the points are the mean and SEM of N=12 animals for each of the three trials on the four training days. The panels on the right indicate the daily average for all trials. (A) Pathlength (total distance travelled before finding the platform). (B) Time in periphery (percentage of the total time spent in an incorrect quadrant). (C) Swim speed.


32

A B

C D

Supplemental Figure 12. Secondary measures of the effects of JNJ-55511118 in the DNMTP assay.

All data are represented as mean ± SEM (N=15), with statistical significance contrast relative to vehicle: *p<0.05, **p<0.01, ***p < 0.001. All tested doses increased the percent of trials omitted, and the two highest doses tested induced a significant increase on latency measures. The magnitudes of these effects were generally modest. (A) Percentage total omissions: omitted trials / total trials started. The analysis used a within-subject design, logistic regression model where the probability of omission is given as a function of treatment, with a random animal effect; statistically significant contrasts relative to vehicle. (B) Sample Press latency: duration from the extension of the sample lever until it is pressed. Within-subject design, repeated-measures ANOVA model comparing the various latencies by dosage group, with a random animal effect. (C) Choice latency: duration from the extension of the levers at choice phase until a selection. Within-subject design, repeated-measures ANOVA model comparing the various latencies by dosage group, with a random animal effect. (D) Collection latency: duration between the delivery of a reward pellet and its collection. Within-subject design, repeated-measures ANOVA model comparing the various latencies by dosage group, with a random animal effect.

The effects of =JNJ55511118on omission percentage in DNMTP

0

20

40

60

80

100

*******

Veh

3 mg/kg10 mg/kg

1 mg/kg

Om

issi

on

per

cen

tag

e (%

)The effects of JNJ55511118

on sample press latency in DNMTP

0

2

4

6

8

****

Veh

3 mg/kg10 mg/kg

1 mg/kg

Sam

ple

pre

ss la

ten

cy (

s)

The effects of JNJ55511118on choice latency in DNMTP

0

2

4

6

***

Veh

3 mg/kg10 mg/kg

1 mg/kg

Ch

oic

e la

ten

cy (

s)

The effects of JNJ55511118on collection latency in DNMTP

0

1

2

3

******

Veh

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A gene code species IN1 TM3 EX2

CCG8_HUMAN Homo sapiens 153 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 184

CCG8_mouse Mus musculus 152 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 183

CCG8_rat Rattus norvegicus 152 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 183

J9NW64_CANFA Canis familiaris 152 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 183

F1RNJ2_PIG Sus scrofa 152 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 183

H2R5V2_PANTR Pan troglodytes 122 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 153

H0XKI8_OTOGA Otolemur garnettii 120 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 151

Q0VFK5_XENTR Xenopus tropicalis 133 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 164

H2STY8_TAKRU Takifugu rubripes 135 KSKRNIILGGGILFVAAGLSNIIGVIVYISAA 166

G3P8Z8_GASAC Gasterosteus aculeatus 145 KSKRNIILGAGILFVAAGLSNIIGVIVYISAA 176

H9GR85_ANOCA Anolis carolinensis 133 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 164

F1MV40_BOVIN Bos taurus 144 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 175

*********.*********************

B gene code species IN1 TM4 EX2

CCG8_HUMAN Homo sapiens 203 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 234

CCG8_mouse Mus musculus 202 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 233

CCG8_rat Rattus norvegicus 202 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 233

J9NW64_CANFA Canis familiaris 202 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 233

F1RNJ2_PIG Sus scrofa 202 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 233

H2R5V2_PANTR Pan troglodytes 172 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 203

H0XKI8_OTOGA Otolemur garnettii 170 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 201

Q0VFK5_XENTR Xenopus tropicalis 183 GWSFYFGGLSFIIAEMIGVLAVNIYIEKNREA 214

H2STY8_TAKRU Takifugu rubripes 185 GWSFYFGGLSFILAEMVGVLAVNIYIEKNKEL 216

G3P8Z8_GASAC Gasterosteus aculeatus 195 GWSFYFGGLSFIMAEMVGVLAVNIYIEKNKEL 226

H9GR85_ANOCA Anolis carolinensis 183 GWSFYFGGLSFILAEMIGVLAVNIYIEKNREA 214

F1MV40_BOVIN Bos taurus 194 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 225

************:**::**********:.:* Supplemental Figure 13. Sequence alignment of TARP-8 across species in the (A) TM3 and (B) TM4

regions. Data and alignments were retrieved from the Uniprot database for genes identified as CACNG8, from a selection of species. Predicted locations for TM3 and TM4 are designated with boxes. The key selectivity residues V177 and G210 (numbering from the human full-length sequence) are highlighted in gray. The transmembrane domains of TARP-8 are very highly conserved across vertebrates, including mammals (human, rat, mouse, monkey, etc.), reptile and amphibian (anole, frog), and fish (fugu, Gasterosteus).


34

A CACNG2 gene code species IN1 TM3 EX2 IN1 TM4 EX2

CCG8_HUMAN Homo sapiens 153 KSKRNIILGAGILFVAAGLSNIIGVIVYISAN 184 203 GWSFYFGGLSFILAEVIGVLAVNIYIERSREA 234

CCG2_HUMAN Homo sapiens 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208

CCG2_MOUSE Mus musculus 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208

CCG2_RAT Rattus norvegicus 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208

J9P237_CANFA Canis familiaris 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208

F1SKJ6_PIG Sus scrofa 62 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 93 110 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 141

H2QLK9_PANTR Pan troglodytes 58 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 89 106 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 137

H0WLV7_OTOGA Otolemur garnettii 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208

A4IIM6_XENTR Xenopus tropicalis 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208

H2RJG2_TAKRU Takifugu rubripes 129 KSRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHRQL 208

G3PNM3_GASAC Gasterosteus aculeatus 129 KSRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIMAEMVGVLAVHMFIDRHRQL 208

G1KSE5_ANOCA Anolis carolinensis 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208

A0JNG9_BOVIN Bos taurus 129 KTRHNIILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIDRHKQL 208

*:::****.***:**:********:******* *******.****:**::*****.::*:* :

B CACNG3 gene code species IN1 TM3 EX2 IN1 TM4 EX2


CCG3_HUMAN Homo sapiens 129 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 160 176 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 207

CCG3_MOUSE Mus musculus 129 RSRHSVILSAGIFFVSAGLSNIIGIIVYISAN 160 176 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 207

CCG3_RAT Rattus norvegicus 129 RSRHSVILSAGIFFVSAGLSNIIGIIVYISAN 160 176 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 207

E2RKC2_CANFA Canis familiaris 72 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 103 119 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 150

F1RFC9_PIG Sus scrofa 72 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 103 119 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 150

H2QAS8_PANTR Pan troglodytes 129 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 160 176 GWSFYFGAFSFIIAQIVGMIAVHIYIEKHQQL 207

H0XZP0_OTOGA Otolemur garnettii 72 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 103 119 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 150

F7BS52_XENTR Xenopus tropicalis 129 KNKHNVILSAGIFFVSAGLSNIIGIIVYISAN 160 177 GWSFYFGALSFIIAEMVGVLAVHMFIEKHRQI 208

CCG3_BOVIN Bos taurus 129 RSRHNVILSAGIFFVSAGLSNIIGIIVYISAN 160 176 GWSFYFGAFSFIIAEIVGVVAVHIYIEKHQQL 207

:.::.:**.***:**:********:******* *******.:***:*:::*::**.::**: ::

C CACNG4 gene code species IN1 TM3 EX2 IN1 TM4 EX2


CCG4_HUMAN Homo sapiens 132 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 163 182 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 213

CCG4_MOUSE Mus musculus 132 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 163 182 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 213

CCG4_RAT Rattus norvegicus 132 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 163 182 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 213

F1RV12_PIG Sus scrofa 132 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 163 182 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 213

H2RDC7_PANTR Pan troglodytes 58 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 89 108 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 139

H0X9H7_OTOGA Otolemur garnettii 128 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 159 178 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 209

F7APU6_XENTR Xenopus tropicalis 133 SRRNNIILSAGILFVAAERRRAGRIEAKKEGE 164 183 GWSFYFGALSFIVAETIGVLXVNIYIERNKEL 214

H9G3D4_ANOCA Anolis carolinensis 113 SSKNNIILSAGILFVAAGLSNIIGIIVYISSN 144 163 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 194

E1BFD7_BOVIN Bos taurus 129 SRKNNIVLSAGILFVAAGLSNIIGIIVYISSN 160 179 GWSFYFGALSFIVAETVGVLAVNIYIEKNKEL 210

. :.**:*.******** . : . ..: *******.****:**.:*** ******:.:*

Supplemental Figure 14. Sequence alignment of TARPs-2, -3, and -4 across species in the TM3

and TM4 regions. Data and alignments were retrieved from the Uniprot database (UniProt Consortium, 2015) for the genes identified as (A) CACNG2, (B) CACNG3, and (C) CACNG4 for the same species as shown in Supplemental Figure 13. For comparison and alignment, TARP-8 is also shown. The transmembrane domains are very highly conserved across vertebrates. In particular, the amino acids corresponding to V177 and G210 in CACNG8 are isoleucine and alanine, respectively, in all other species examined.

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