ORIGINAL ARTICLE

High- and low-affinity sites for sodium in δ-OR-Gi1α(Cys351-Ile351) fusion protein stably expressed in HEK293 cells;functional significance and correlation with biophysical stateof plasma membrane

Miroslava Vošahlíková & Piotr Jurkiewicz &

Lenka Roubalová & Martin Hof & Petr Svoboda

Received: 15 October 2013 /Accepted: 10 February 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The effect of sodium, potassium, and lithium on δ-opioid receptor ligand binding parameters and coupling withthe cognate G proteins was compared in model HEK293 cellline stably expressing PTX-insensitive δ-OR-Gi1α (Cys351-Ile351) fusion protein. Agonist [3H]DADLE binding was de-creased in the order Na+≫Li+>K+>(+)NMDG. When plottedas a function of increasing NaCl concentrations, the bindingwas best-fitted with a two-phase exponential decay consider-ing two Na+-responsive sites (r2=0.99). High-affinity Na+-sites were characterized by Kd=7.9 mM and represented 25%of the basal level determined in the absence of ions. Theremaining 75 % represented the low-affinity sites (Kd=463 mM). Inhibition of [3H]DADLE binding by lithium,potassium, and (+)-NMDG proceeded in low-affinity manneronly. Surprisingly, the affinity/potency of DADLE-stimulated[35S]GTPγS binding was increased in a reverse order: Na+<K+<Li+. This result was demonstrated in PTX-treated as wellas PTX-untreated cells. Therefore, it is not restricted toGi1α(Cys

351-Ile351) within the δ-OR-Gi1α fusion protein,but is also valid for stimulation of endogenous G proteins ofGi/Go family in HEK293 cells. Biophysical studies of inter-action of ions with polar head-group region of lipids usingLaurdan generalized polarization indicated the low-affinitytype of interaction only proceeding in the order: Cs+<K+<Na+<Li+. The results are discussed in terms of interaction ofNa+, K+ and Li+ with the high- and low-affinity sites located in

water-accessible part of δ-OR binding pocket. We also con-sider the role of negatively charged Cl−, Br−, and I− counteranions in inhibition of both [3H]DADLE and [35S]GTPγSbinding.

Keywords δ-opioid receptor .Monovalent ions . Agonist andantagonist binding . [35S]GTPγS binding .Membranebiophysics . Laurdan fluorescence

Abbreviationsa.a. amino acidAC adenylyl cyclaseβ-AR β-adrenergic receptorDADLE (2-D-alanine-5-D-leucine)-

enkephalin=Tyr-D-Ala-Gly-Phe-D-Leuδ-OR δ-opioid receptorδ-OR-Gi1α cells HEK393 cells stably

expressing δ-OR-Gi1α (C351-I351)fusion protein

DMEM Dulbecco’s modified eagle’s mediumDPH 1,6-diphenyl-1,3,5-hexatrieneGPCR G protein-coupled receptorG proteins heterotrimeric guanine nucleotide-

binding regulatory proteinsGi/Goα G proteins inhibiting adenylyl

cyclase activity in pertussis toxinsensitive manner

[35S]GTPγS guanosine-5′-O-[γ-35S]-triphosphateHEK human embryonic kidneyκ-OR κ-opioid receptorLUV large unilamellar vesiclesSUV small unilamellar vesiclesLaurdan 6-dodecanoyl-2-

dimethylaminonaphthalene

M. Vošahlíková : L. Roubalová : P. Svoboda (*)Institute of Physiology, Academy of Sciences of the Czech Republicv.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republice-mail: svobodap@biomed.cas.cz

P. Jurkiewicz :M. HofJ. Heyrovský Institute of Physical Chemistry, Academy of Sciencesof the Czech Republic, v.v.i., Dolejškova 3, 18223 Prague 8, CzechRepublic

Naunyn-Schmiedeberg's Arch PharmacolDOI 10.1007/s00210-014-0962-8

Laurdan GP Laurdan generalized polarizationGPex (av) average of GPex values between

320 nm and 400 nmμ-OR μ-opioid receptor(+)-NMDG N-methyl-D-glucamineOR opioid receptorPC phosphatidylcholinePOPC 1,2-palmitoyl-oleoyl-sn-

glycero-3-phosphocholinePOPS 1,2-palmitoyl-oleoyl-sn-

glycero-3-phosphoserinePS phosphatidylserinePTX pertussis toxinSM sphingomyelinSR solvent relaxation techniqueTM transmembraneλex excitation wavelengthλem emission wavelength

Introduction

Interaction of monovalent ions with opioid receptors

The interaction of monovalent cations with signaling cascadesinitiated by opioid receptors (OR) was studied since the veryearly days of GPCR-oriented research. The stereo-specificbinding of OR agonists [3H]dihydromorphine and[3H]oxymorphine to brain membranes was found to beinhibited by increasing concentrations of NaCl and to a lessextent by LiCl (Pert and Snyder 1974). Other ions, potassium,rubidium, and cesium were not effective (Na+≫Li+>K+≈Rb+≈Cs+). On the contrary, antagonist binding to OR wasaffected in a positive manner. Binding of the antagonist[3H]naloxone was increased, again in the order Na+≫Li+>K+≈Rb+≈Cs+.

Original data of Pert and Snyder (Pert and Snyder 1974)were confirmed and extended by studies on membranes pre-pared from the whole guinea pig brain or cerebellum (Patersonet al. 1986). Binding of μ-OR agonist [3H]DAMGO wasinhibited by monovalent ions in the order: NaCl>LiCl>NH4Cl≥KCl; antagonist [3H]naloxone binding was increased,again with a strong preference for sodium. Similar data wereobtained for δ-OR by analysis of binding of specific agonists[3H]DPDPE and [3H]DADLE.

Later studies of interaction of sodium cations with δ-ORusing the known amino acid sequence of δ-OR indicated that aconserved aspartic acid 95 in the second TM region is criticalfor Na+ regulation of agonist binding (Kong et al. 1993).Recent data based on the crystal structure of the μ-OR in acomplex with the irreversible antagonist morphinan (Mangliket al. 2012) and mouse δ-OR with the antagonist naltrindol

(Granier et al. 2012) at 2.8–3.3 Å resolution indicated thatthese antagonists bind within a large solvent-exposed pocket.This extra large “water-accessible space” of the antagonistbinding site of OR is a characteristic feature of this type ofreceptors and differs from most other GPCRs including β2-AR (Filizola and Devi 2012). Furthermore, the binding pocketof opioid receptors can be divided into two distinct regions.The lower/bottom or inner part of this pocket is highly con-served among OR whereas the upper part (exposed to extra-cellular aqueous space) contains divergent residues that confersubtype selectivity.

The function of GPCR relies on specific lipid environmentand often strongly depends on the presence of cholesterol andions (Burger et al. 2000; Gao and Ijzerman 2000; Goblyos andIjzerman 2011; Brejchova et al. 2011). The understanding ofthe significance of these small molecules for GPCR functionhas been extended by structural analysis of human A2A aden-osine receptor (A2AAR) containing an apocytochrome b562 ina complex with a subtype-selective antagonist at 1.8 Å reso-lution (Liu et al. 2012). The high resolution of the structureof this chimeric protein made it possible to identify acomprehensive network of 57 internal water molecules, ahighly conserved binding site for a sodium ion, 3 cho-lesterol molecules, and 23 lipid acyl chains bound to thereceptor.

The association of GPCRs and G proteins with the plasmamembrane makes them susceptible to their lipid environmentso that lipid-protein interactions are important for their func-tion (Anderson and Jacobson 2002). Differentiated domainswith specific protein and lipid composition exist in plasmamembranes of manymammalian cells such as basal and apicalepithelial/endothelial cells and neurons. Membrane domains/lipid rafts or caveolae were found to be enriched in cholester-ol, specific lipids such as sphingomyelin and G proteins (Pike2004; Allen et al. 2007; Chini and Parenti 2009). More spe-cifically, κ-OR were found to be localized in lipid rafts (Xuet al. 2006) and analysis of μ-OR- and δ-OR-G protein cou-pling indicated the compartmentation of these signaling mol-ecules in PM (Remmers et al. 2000). Cholesterol reduction bymethyl-β-cyclodextrin attenuated δ-OR-initiated signaling inneuronal cells (Huang et al. 2007) and in HEK293 cells stablyexpressing δ-OR-Gi1α fusion protein (Brejchova et al. 2011).Adenylyl cyclase super-activation induced by prolonged ex-posure to morphine was found to be dependent on receptorlocalized within lipid rafts and independent of receptor inter-nalization (Zhao et al. 2006). Palmitoylation and membranecholesterol stabilized the homooligomerization of μ-OR andcoupling with G proteins (Zheng et al. 2012).

Experiments based on plasmon-waveguide resonancespectroscopy (Alves et al. 2005) indicated that the unbound,inactive δ-OR receptors were enriched in POPC-rich domainof lipid bilayer composed of 1:1 mixture of POPC and SM.The agonist- and antagonist-bound receptors were

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concentrated in SM-rich component (with a twofold greaterpropensity in the case of the agonist) characterized by a greaterthickness. Since it is known that receptor activation involveschanges in the orientation of trans-membrane helices with anincrease in receptor vertical length, i.e., perpendicular to theplane of the plasma membrane (Salamon et al. 2000), it washypothesized that the driving force for receptor redistributioncomes from the increased hydrophobic match of the elongatedreceptor for the thicker SM-enriched part of the bilayer, asorting mechanism that has previously been observed in othersubcellular compartments (Jensen and Mouritsen 2004; Lee2004). Thus, the studies of model lipid membranes composedfrom defined lipids brought an important advance in under-standing of OR-G protein coupling.

Literature data mentioned above justify the efforts combin-ing biophysical analysis of model and natural lipid mem-branes with biochemical characterization of OR function innatural lipid environment. The specific, high-affinity interac-tion of monovalent cations with allosteric site of ligand bind-ing pocket of OR (characterized by strong preference forsodium; Manglik et al. 2012; Granier et al. 2012; Fenaltiet al. 2014) should overlap “somewhere at receptor-lipidinterfaces” with the less specific and subsequently non-specific interaction of these ions with lipid molecules. It isreasonable to assume that all these interactions proceed inclose association with reorganization of water molecules inand around receptor molecules.

Interaction of ions with model and natural lipid membranes

The influence of physiologically relevant ions (Na+, K+, Cl−,Ca2+, and Mg2+) on model lipid membranes was studiedextensively in the past (Bockmann et al. 2003; Vacha et al.2009). Other ions, such as Li+, Cs+, NH4

+, Ba2+, La3+, F−, Br−,I−, NO3

−, and SCN−, were also investigated (Garcia-Celmaet al. 2007; McLaughlin et al. 1978). The specificity of theionic effects is known to be more pronounced for anions thanfor cations. Therefore, experimental data on cationic effectswere sparser (Clarke and Lupfert 1999; Garcia-Celma et al.2007; Kunz et al. 2004; Tatulian 1987). The strongest were theeffects of multivalent and monovalent cations characterizedby a high charge density (Mg2+, Ca2+, Li+; Faraday 2013;Pabst et al. 2007).

Since some of naturally occurring lipids are anionic, ad-sorption of cations to natural lipid membranes is stronger thanto electrically neutral model membranes. This is due to thecoulombic attraction between positively charged cations andnegatively charged head-groups in the inner leaflet of naturallipid bilayer. For example, in mixed POPC/POPS (4:1, mol/mol) lipid bilayers, which are often used as a model of theinner leaflet of a plasma membrane enriched in PS, adsorptionof cations was found to follow the reversed Hofmeister series:Na+>K+>Cs+ (Eisenberg et al. 1979). Computer simulations

showed that adsorption of Na+ to negatively charged mem-branes often leads to complexing of up to 4 lipid molecules,but the exact binding sites were dependent on the force field(Broemstrup and Reuter 2012; Pandit et al. 2003; Porasso andLopez Cascales 2009). In our previous studies, interaction ofNa+, K+, and Cs+ with model lipid membranes consisting of aPOPC/POPS mixture was investigated by the solvent relaxa-tion technique (SR). SR showed a significant decrease ofhydration and mobility of lipid carbonyls. This was indicatedby the measurement of Laurdan general polarization whichdecreased in the order Na+>K+>Cs+ (Jurkiewicz et al. 2012).

With the aim to analyze the effect of monovalent cations onδ-opioid receptor in more details (Vosahlikova and Svoboda2011) and distinguish the possible differences between indi-vidual ions when interacting with the polar membrane–waterinterface and the hydrophobic interior of membrane bilayer,plasma-membrane-enriched fraction was prepared fromHEK293 cell stably expressing δ-OR-Gi1α (C351-I351) hybridprotein (Bourova et al. 2009; Moon et al. 2001a). These(natural) membranes were compared with model lipid bilayersrepresented by negatively charged POPC/POPS andPOPC/POPS/cholesterol lipid vesicles. Laurdan generalizedpolarization was applied for the analysis of interaction ofdifferent monovalent cations with both types of membranes.δ-OR-Gi1α (C351-I351) cells represent a useful experimentaltool (Bourova et al. 2003; Brejchova et al. 2011), as thecovalent bond between δ-OR and Gi1α (C351-I351) providesthe permanent and fixed, 1:1 stoichiometry and C351-I351

mutation provides resistance to PTX together with an extraor-dinarily high efficacy of coupling between δ-OR and Gi1α(C351-I351) protein (Moon et al. 2001a, b).

Materials and methods

Materials

δ-OR agonist [3H]DADLE [enkephalin (2-D-alanine-5-D-leucine), 50 Ci/mmol, NET 648] was from Perkin-Elmer,NEN Life Science; DOR antagonist [3H]naltrinol (60 mCi/mmol) was from BioTrend, Gmbh. [35S]GTPγS (1,250 Ci/mm o l ) w a s p u r c h a s e d f r o m P e r k i n E l m e r(NEG030H001MC); δ-OR agonist DADLE [(2-D-alanine-5-D-leucine)-enkephalin=Tyr-D-Ala-Gly-Phe-D-Leu] (E7131)and N-methyl-D-glucamine (M2004) was from Sigma(E7384). Complete protease inhibitor cocktail was fromRoche Diagnostic, Mannheim, Germany (cat. no. 1697498).

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC) and 1-palmi toyl -2-oleoyl -sn -g lycero-3-phosphoserine (POPS) were obtained from Avanti PolarLipids (Alabaster, AL, USA). The fluorescence probes 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) and

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1,6-diphenyl-1,3,5-hexatriene (DPH) were purchased fromMolecular Probes (Eugene, OR, USA).

LiCl with purity ≥99 and KCl with purity ≥98 were obtain-ed from Fluka (Buchs, Switzerland), NaCl and CsCl both withpurity >99 %, NaOH, and ethylenediaminetetraacetic acid(EDTA) were from Sigma Aldrich (St. Louis, USA). Saltsand EDTA were dissolved in Mili Q (Milipore, USA) water.Organic solvents of spectroscopic grade were supplied byMerck (Darmstadt, Germany).

Cell culture

HEK293 cells stably expressing δ-OR-Gi1α (C351-I351) fusionprotein were cultivated in DMEM (Sigma) supplemented with4 mM (0.584 g/l) L-glutamine and 10 % v/v newborn calfserum at 37 °C. Geneticin (800 μg/ml) was included in thecourse of cell cultivation. The cells were grown to 60–80 %confluency, treated for 16 h with pertussis toxin (25 ng/ml)and harvested by centrifugation for 10 min at 1,800 rpm(300×g). The cell sediment was frozen in liquid nitrogen andstored at −80 °C until use.

Membrane preparation

Plasma membrane-containing P2 particulate fraction was pre-pared from cell sediments stored at -80 °C as described beforeby Wise et. al. (Wise et al. 1997). Cell pellets were re-suspended in 0.5 ml of 10 mM Tris–HCl, 0.1 mM EDTA,pH 7.5 (TE buffer) and rupture of the cells was achieved with50 strokes in a hand-held Teflon-glass homogenizer followedby passage (10 times) through a 25-gauge needle. Cell lysateswere centrifuged at 1,000×g for 10 min in a Beckman TJ-6centrifuge to pellet the nuclei and unbroken cells and P2particulate fraction was then recovered by centrifugation ofthe supernatant at 200,000×g for 30min in a Beckman TL 100bench-top ultracentrifuge using a Beckman TLA 100.2 rotor.Membrane sediment was re-suspended in TE buffer, frozen inliquid nitrogen and stored at −80 °C.

[3H]DADLE binding

Membranes (20 μg per tube) were incubated in a finalvolume of 0.1 ml of binding mix containing 15 nM[3H]DADLE, 50 mM Tris–HCl, 10 mM MgCl2, 1 mMEDTA (pH 7.4)±increasing concentrations of NaCl, KCl,or LiCl for 60 min at 30 °C. Non-specific binding wasdetermined in the presence of 10 μM DADLE. Separationof bound and free radioactivity was performed by filtrationthrough Whatman GF/B filters in Brandel cell harvester, thefilters were washed 3× with 3 ml of ice-cold incubationbuffer and radioactivity was determined by liquid scintilla-tion. Data were analyzed by GraphPad Prism 4.

[3H]naltrindol binding

Membranes (20 μg protein) were incubated with δ-OR antag-onist [3H]naltrindol (5 nM) in 0.1 ml of 75 mM Tris–HCl,pH 7.4, 12.5 mMMgCl2*, 1 mM EDTA for 60 min at 30 °C.The bound and free radioactivity was separated by filtrationthrough Whatman GF/B filters. The filters were washed 3×with 3 ml of ice-cold incubation buffer and placed in 4 ml ofscintillation cocktail (Rotizscint). Radioactivity was deter-mined after 60 min at laboratory temperature in the dark.The non-specific binding was determined as that remainingin the presence of 10 μM non-radioactive naltrindol. Datawere calculated by GraphPadPrism 4.

*, the number represents the total MgCl2 concentration inreactionmix; for calculation of free magnesium concentration,see Higashijima et. al. (Higashijima et al. 1987a, b, c).

Agonist-stimulated [35S]GTPγS binding

Membranes (20 μg protein per assay) were incubated with(total) or without (basal) 1 μM DADLE in a final volume of100 μl of reaction mix containing 20 mM HEPES, pH 7.4,3 mMMgCl2, 2 μMGDP, 0.2 mM ascorbate and [35S]GTPγS(1–2 nM) for 30 min at 30 °C. The binding reaction wasdiscontinued by dilution with 3 ml of ice-cold 2 mMHEPES, pH 7.4, 0.15 mM MgCl2 and immediate filtrationthrough Whatman GF/C filters on Brandel cell harvester.Radioactivity remaining on the filters was determined byliquid scintillation. When analyzing the dose–responsecurves, DADLE concentration was increased from 2×10−9

to 1×10−6 M. Non-specific GTPγS binding was determinedin parallel assays containing 10 μMGTPγS. The binding datawere analyzed by GraphPad Prism 4; EC50 and Bmax valueswere calculated by fitting the data with rectangular hyperbola.

Isolation of plasma membrane fraction in PercollR densitygradient

δ-OR-Gi1α-HEK293 cells were cultivated in 15 flasks(80 cm2), washed in PBS, and harvested by centrifugationfor 10 min at 1,800 rpm (300×g) in 50 ml conical vials. Thecell pellet was homogenized in 250mM sucrose, 20mMTris–HCl, 3 mM MgCl2, 1 mM EDTA, pH 7.6 plus fresh 1 mMPMSF and a complete protease inhibitor cocktail (STEMmedium) in loosely fitting, Teflon-glass homogenizer for7 min at 1,800 rpm. The cell homogenate was centrifugedfor 7 min at 3500 rpm (1,160×g) to separate cell debris andnuclear fraction (remaining in the sediment) from post-nuclearsupernatant, PNS. The 3 ml of PNS was applied on the top of20 ml of 30 % v/v PercollR in thick polycarbonate BeckmanTi70 tubes. Centrifugation for 35 min at 30,000 rpm(66,200×g) resulted in separation of two clearly visible layers.The upper layer represented the plasma membrane fraction

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(PM), the lower layer represented mitochondria. The upperlayer was diluted 1:4 in distilled water and centrifuged inBeckman Ti70 rotor for 90 min at 50,000 rpm (184,000×g).The PM sediment was removed from the compact, gel-likesediment of PercollR, re-homogenized by hand in a smallvolume of 50 mM Tris–HCl, 1 mM EDTA, pH 7.4 (TEmedium), frozen in liquid nitrogen and stored at −80ºC. Thistype of membranes was used in fluorescence spectroscopystudies as the P2 particulate membrane fraction exhibited aspontaneous aggregation in the cuvette.

Steady-state fluorescence anisotropy of DPH

PercollR-purified PM were labeled with DPH by the fastaddition (under mixing) of 1 mM DPH in freshly distilledacetone to the membrane suspension (0.1 mg protein/ml;1 μM final concentration); after 30 min at 25 °C, which wereallowed to ensure the optimum incorporation of the probe intothe membrane interior, the anisotropy of DPH fluorescence(rDPH) was measured at Ex 365 nm/Em 425 nm wavelengths.Under these conditions, the fluorescence intensity of themembrane-bound DPH was≈500× higher than that of theunbound, free probe in aqueous medium; light scatteringproblems could be therefore omitted. Steady-state fluores-cence anisotropy, rDPH, was calculated by the formula:rDPH=(Ivv− Ivh)/(Ivv+2Ivh) according to Shinitzky andBarenholz (1978) and Shinitzky (1984).

Laurdan generalized polarization in HEK293 cell membranes

Incorporation of the Laurdan fluorescent probe into isolatedHEK293 cell membranes (PercollR-purified) was performedby adding of 1 mM stock solution in methanol to membranesuspension (10 μM final concentration) followed by incuba-tion for 30min at 40 °C under mixing. Recording of excitationspectra was carried out at 20 °C and 40 °C. The excitationspectra were recorded at the emission wavelengths set to440 nm and 490 nm in a 300–430 nm range of excitationwavelengths, λex. Subsequently, the values of excitation gen-eralized polarization (GPex) were calculated from intensitiesof Laurdan fluorescence in themost sensitive range of λex, i.e.,between 320 and 400 nm, according to Parasassi et al.(Parasassi et al. 1991, 1994). The excitation GPwas calculatedaccording to the equation:

GPex λexð Þ ¼ I440 − I490I440 þ I490

ð1Þ

where I490 and I440 represent fluorescence intensities detectedat 490 nm and 440 nm, respectively, excited at excitationwavelength λex.

Laurdan generalized polarization in large unilamellarPOPC/POPS vesicles

Chloroform solutions of POPC and POPS in a 4:1 molar ratiowere mixed with methanol solution of Laurdan (1:100 mol/mol dye to lipid). The solvents were evaporated under thestream of nitrogen while heated, and left in the vacuum for atleast 2 h in order to remove the remaining organic solvents.Dry lipid film was suspended in 1.5 ml of 1,000 mM saltsolutions with 0.1 mM EDTA or in 0.1 mM EDTA alone (nosalt) and vortexed for 4 min. EDTA was used to chelatecalcium ions dissolved from the glassware. Large unilamellarvesicles (LUV) were formed by extrusion through a polycar-bonate membrane (Avestin, Ottawa, Canada) with 100 nmpores. The prepared samples were transferred to a 1 cm quartzcuvette. The final concentration of phospholipids in the cu-vette was 1 mM.

Laurdan generalized polarization in small unilamellarPOPC/POPS vesicles

The procedure used for preparation of small unilamellar ves-icles was the same as that described in the previous paragraphwith a few exceptions. The 800 mM salt and 0.5 mM EDTAsolutions were used. Instead of extrusion, small unilamellarvesicles (SUV) were formed by sonication of the lipid sus-pension for 5 min using a tip sonicator (Sonopuls HD 2,070,Bandelin electronic GmbH & Co. KG, Berlin, Germany) at10 % power under cooling in a water bath at room tempera-ture. The final concentration of phospholipids in the cuvettewas 0.9 mM.

Laurdan generalized polarization in large unilamellarPOPC/POPS/cholesterol vesicles

Chloroform solutions of POPC (46 mol%), cholesterol(33 mol%), and POPS (20 mol%) were mixed with methanolsolution of Laurdan (1:100 mol/mol dye to lipid), the solventswere evaporated under the stream of nitrogen while heatedand left in the vacuum for at least 2 h in order to remove theremaining organic solvents. Dry lipid film was suspended in1.5 ml of 800 mM salt solutions with 0.1 mM EDTA or in0.1 mM EDTA alone (no salt). All subsequent steps wereidentical with the preparation of POPC/POPS LUV.

Measurement of steady-state Laurdan fuorescencein δ-OR-Gi1α-HEK293 cell membranes and vesicularmembrane bilayers

Steady-state fluorescence spectra of Laurdan were recordedusing Fluorolog-3 spectrofluorimeter (model FL3–11; JobinYvon Inc., Edison, NJ, USA). Temperature was maintainedwithin ±0.5 °C using a water-circulating bath.

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Miscellaneous

Protein was determined by the Lowry method using bovineserum albumin as standard. Statistical analysis and fitting ofthe experimental data with mathematical functions were per-formed by GraphPad Prism 4.

Results

Agonist and antagonist binding to δ-OR-Gi1α (C351-I351)

The effect of increasing concentrations of monovalent cationsNa+, K+, Li+, and (+)-NMDG on the binding of δ-OR agonist[3H]DADLE was analyzed in membranes isolated from PTX-treated δ-OR-Gi1α (C351-I351) cells at equimolar concentra-tion of chloride anion. Data presented in Fig. 1 indicated thatthe inhibitory effect of sodium was much stronger andproceeded with higher affinity (Fig. 1a) than that of lithium,potassium, and (+)-NMDG (Fig. 1c, b, d). The high, basallevel of binding in the absence of sodium (1.58 pmol×mg−1,100 %) was decreased to 65±5 % in 100 mMNaCl (p<0.01);at this concentration, the inhibitory effect of LiCl, KCl, and (+)-NMDG was undetectable, i.e., [3H]DADLE binding in thepresence of these cations was not significantly different fromthe basal level (p>0.05). Addition of 200 mM NaCl or LiClresulted in a highly significant decrease of binding to 55 %(p<0.001) and 86 % (p<0.05) of the basal level, respectively,

but the effect of K+ and (+)-NMDG was non-significant. It isworth noticing that at 145 mMNa+, i.e., sodium concentrationidentical with that in extracellular milieu, [3H]DADLE bind-ing represented 59 % of the basal level. Thus, in the low-concentration range, monovalent ions inhibited agonist bind-ing to δ-OR-Gi1α in the order Na+≫Li+>K+>(+)-NMDG(Fig. 1a–d).

The high range of ion concentrations (>200 mM) wascharacterized by further decrease of [3H]DADLE binding.When analyzing the inhibition curve in the whole range ofconcentrations by GraphPad Prism 4, the data were best-fittedwith a two-phase exponential decay considering two Na+-responsive sites (r2=0.99). This was evidenced by the biphas-ic inhibition curve presented in Fig. 1a. The high-affinity siteswere characterized by the dissociation constant Kd=7.9 mMand represented 25 % (0.314 pmol×mg−1) of the basal leveldetermined in the absence of ions, 100%. The remaining 75%represented the low-affinity sites (Kd=463 mM). Half-maximum inhibition of [3H]DADLE binding elicited byhigh- and low-affinity sites was observed at 5.4 mM and321 mM NaCl, respectively.

The inhibition of agonist binding by lithium (Fig. 1c), potas-sium (Fig. 1b), and (+)-NMDG (Fig. 1d) proceeded only in alow-affinity manner and reached the low-bottom level at 32 %(Li+), 57 % (K+) and 89 % [(+)-NMDG] of the basal level,respectively. Half-maximum inhibition was observed at355 mM (Li+), 450 mM (K+) and 650 mM [(+)-NMDG]concentration of these ions. Thus, the order of sensitivity

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Fig. 1 Inhibition of agonist[3H]DADLE binding to DOR-Gi1α protein by monovalent ions.Membranes were incubated with15 nM [3H]DADLE in thepresence of increasingconcentrations of NaCl (a), KCl(b), LiCl (c), or (+)-NMDG (d).Specific binding was determinedas described in the “Materials andmethods” section, expressed aspmol per mg of membrane proteinand analyzed by GraphPadPrizm4.Binding in the absence of ions(1.58 pmol×mg−1) was regardedas the basal level, 100 %; bindingat 800 mM concentration ofmonovalent ions was regarded asthe low-bottom level. Inhibitioncurve was best-fitted with a two-phase exponential decay model inthe case of NaCl or withpolynomials of higher order in thecase of KCl, LiCl, and (+)-NMDG.Data represent the means±SEM of3 binding assays performed intriplicate

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Na+>Li+>K+>(+)-NMDG was noticed even for the low-affinity type of interaction of ions with the agonist binding site(Fig. 1a–d).

The ability of sodium to distinguish between agonist andantagonist binding was clearly demonstrated in δ-OR-Gi1α(C351-I351) membranes isolated from PTX-treated cells as anincrease of [3H]natrindol binding (Fig. 2). In sodium-containing media (Fig. 2a), the basal level of binding (2.24pmol×mg−1; 100 %) was increased to 124 % (100 mM) and131 % (200 mM) NaCl, respectively; half-maximum stimula-tion was observed in 30 mMNaCl. This ability was shared bylithium (Fig. 2c) and potassium (Fig. 2b), but it was manifest-ed in low-affinity manner only since the first significant in-crease of binding was observed at concentrations higher than150 mM (LiCl) or 200 mM (KCl). Half-maximal increase of[3H]natrindol binding was detected at 160 mM LiCl and215 mMKCl, respectively. The increase of antagonist bindingby (+)-NMDG (Fig. 2d) was very low and not significantlydifferent from the basal level even at 800 mM concentration(113 %). Similar data were obtained in membranes preparedfrom PTX-untreated cells (data not shown).

Thus, the δ-OR expressed in the fusion protein with Gi1αbehaves in a similar way as the wild type δ-OR in other cellenvironments containing endogenous G proteins of Gi/Go

family. δ-OR-Gi1α (C351-I351) is endowed with the ability to

distinguish between agonist and antagonist binding in theorder Na+>K+≈Li+>(+)-NMDG (Fig. 2a–d).

Intrinsic efficacy and potency/affinity of δ-OR-Gi1α(C351-I35) in different ion media

In the second part of our work, the effect of monovalent cationson Gi1α (C351-I35) activation by DADLE was measured byhigh-affinity [35S]GTPγS binding assays in membranes pre-pared from PTX-treated cells. A high level of binding wasdetected in the absence of ions and this level was not affectedby the increasing concentrations of DADLE. Addition of mono-valent ions at 200 mM concentration resulted in a large inhibi-tion of the binding: from 0.622 pmol×mg−1 to 0.143 (NaCl),0.241 (KCl), and 0.209 pmol×mg−1 (LiCl), respectively(Table 1A). This inhibition was reversed by increasing concen-trations of DADLE (10−9–10−6 M). Dose–response curves ofDADLE in 200 mM NaCl were characterized by the highestmaximum of response (350 %) followed by LiCl (231 %) andKCl (216 %). Sodium cations were thus more efficient thanlithium and potassium in providing a proper ionic milieu foractivation of δ-OR-Gi1α protein by an agonist; in other words,preferential sensitivity of δ-OR-Gi1α to sodium was also man-ifested for intrinsic efficacy of δ-OR in the order Na+>Li+≈K+.

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Naunyn-Schmiedeberg's Arch Pharmacol

A reverse order was found for the potency of DADLE-response. Surprisingly, the potency/affinity of DADLE-response of Gi1α protein, quantitatively characterized by(EC50)

−1 values was increased in the order of: Na+ (1.961×107 M−1; EC50=5.1×10

−8 M)<K+ (1.042×108 M−1; EC50=9.6×10−9 M)<Li+ (1.852×108 M−1; EC50=5.4×10

−9 M);(Table 1A).

Similar data were obtained in membranes isolatedfrom PTX-untreated cells (Table 1B), both in terms ofefficacy and potency of DADLE response. The δ-ORagonist had no effect on [35S]GTPγS binding in theabsence of ions, but the low level of binding in thepresence of ions was increased by increasing concentra-tions of DADLE with maximum response in 200 mM

sodium chloride (327 %), followed by lithium (248 %)and potassium (237 %). As before, the potency/affinityof DADLE response was increased in a reverse order:Na+ (1.538×107 M−1; EC50=6.5×10

−8 M)<K+ (5.000×107 M−1; EC50=2.0×10

−8 M)<Li+ (1.190×108 M−1;EC50=8.4×10

−9 M). Thus, the maximum response ofGi1α protein (in PTX-treated cells) or of Gi1α proteinplus endogenous G proteins of Gi/Go family (in PTX-untreated cells) was highest in sodium (Na+>Li+≈K+),while the potency/affinity of G protein response in-creased in the order Na+<K+<Li+.

Subsequently, a completely independent set of mem-branes prepared from different cell cultures was used forcomparing the dose–response curves in 100 mM and

Table 1 DADLE-stimulated [35S]GTPγS binding in membranes prepared from PTX-treated and PTX-untreated δ-OR-Gi1α-HEK293 cells; the effectof 200 mM NaCl, KCl, and LiC

A PTX-treated

EC50 % Bbasal Bmax Δmax

NaCl 5.1×10-8 M 350 0.143 0.499 0.356 ** ** ** * **

KCl 9.6×10-9 M ** 216 ** 0.241 ** 0.520 NS 0.279 *** NS NS ** NS

LiCl 5.4×10-9 M 231 0.209 0.481 0.272

B PTX-untreated

EC50 % Bbasal Bmax Δmax

NaCl 6.5×10-8 M 327 0.178 0.582 0.404** * ** * **

KCl 2.0×10-8 M ** 237 * 0.222 ** 0.526 * 0.304 *** NS NS NS NS

LiCl 8.4×10-9 M 248 0.211 0.523 0.312

[35 S]GTPγS binding was measured in P2 membrane fraction isolated from PTX-treated (A) or PTX-untreated cells (B) as described in the “Materialsand methods” section. Binding assays were performed in 200 mM NaCl, KCl, or LiCl. EC50 (M) and Bmax (pmol × mg−1 ) values were calculated byGraphPadPrizm4. Bmax values were also expressed as the ratio (%) between the maximum DADLE-stimulated (Bmax) and the basal level (Bbasal) ofbinding. Net increment of agonist stimulation (Δmax) was calculated as the difference between Bmax and Bbasal values. Numbers represent the means±SEM of 3 binding assays, each performed in triplicate. Data were analyzed by one-way ANOVA followed by Neuman–Keuls post test (*p<0.05,**p<0.01, NS non-significant). (A) In PTX-treated membranes, [35 S]GTPγS binding in the absence of ions was 0.622 pmol×mg−1 and this level wasdecreased to 0.143 (NaCl), 0.241 (KCl), and 0.209 (LiCl)pmol×mg−1 by the addition of 200mMNaCl, KCl, or LiCl, respectively. (B) In PTX-untreatedmembranes, [35 S]GTPγS binding in the absence of ions was 0.809 pmol×mg−1 and this level was decreased to 0.178 (NaCl), 0.222 (KCl), and 0.211(LiCl)pmol×mg−1 by the addition of 200 mM NaCl, KCl, or LiCl, respectively

Naunyn-Schmiedeberg's Arch Pharmacol

200 mM NaCl. Maximum DADLE response determinedin 100 mM NaCl, KCl and LiCl (Fig. 3, left panels)decreased with the order of: 335 % (NaCl)>171 %(LiCl)≈162 % (KCl), respectively; the potency/affinityof DADLE response was increased in the reverse order:Na+ (1.031×108 M−1; EC50=9.7×10

−9 M)<K+ (1.754×108 M−1; EC50=5.7×10

−9 M)<Li+ (3.125×108 M−1;EC50=3.2×10

−9 M). Repetition of binding assays at200 mM NaCl, KCl, and LiCl with these new mem-branes resulted in the same orders of efficacy andpotency/affinity as in 100 mM NaCl (Fig. 3, rightpanels). The maximum response of Gi1α in the mem-branes prepared from PTX-treated δ-OR-Gi1α cells wasdecreased with the sequence: 438 % (NaCl)>298 %

(LiCl)≈271 % (KCl); the potency/affinity was increasedin a reverse order: Na+ (2.500×107 M−1; EC50=4.0×10−8 M)<K+ (1.163×108 M−1; EC50=8.6×10

−9 M)<Li+

(1.961×108 M−1; EC50=5.1×10−9 M).

Data presented in Fig. 3 also indicated that (a)DADLE was unable to increase the high basal level of[35S]GTPγS binding in the absence of ions even at1 μM concentration and (b) sodium was the most effi-cient ion in inhibiting this high level of binding. Asexpected, inhibitory effect at 200 mM salt concentrationwas stronger than at 100 mM NaCl, LiCl and KCl;inhibition decreased in order NaCl>LiCl≈KCl. Thes am e o r d e r w a s d e t e c t e d i n h i g h - a f f i n i t y[α-32P]GTPase assays (data not shown).

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Fig. 3 Comparison of dose–response curves of DADLE-stimulated [35S]GTPγS bindingin 100 mM and 200 mM NaCl,KCl, and LiCl. [35S]GTPγSbinding was measured in theabsence (Bbasal) or presence(BDADLE) of increasingconcentrations of DADLE (2×10−9–1×10−6 M) in membranesprepared from PTX-treated cells.Binding assays were performed inthe absence (square) or presence(filled square) of 100 mM (leftpanels) or 200 mM (right panels)concentration of monovalentcations (NaCl, KCl, and LiCl).EC50 and Bmax values werecalculated by GraphPadPrizm4.Data represent the means±SEMof 3 binding assays eachperformed in triplicate

Naunyn-Schmiedeberg's Arch Pharmacol

Fluorescence studies of Laurdan generalized polarizationin δ-OR-Gi1α-HEK293 membranes

In the third part of our work, we tried to find some explanationfor the existence of high- and low-affinity Na+-responsivesites in δ-OR-Gi1α membranes. The high-affinity sites wereeasy to understand as the specific, high-affinity sodium effecton agonist binding to δ-OR proceeds as an interaction ofpositively charged Na+ with negatively charged carboxylgroup of aspartic acid 95 (Kong et al. 1993b). The chargeneutral mutation of Asp 95 for Arg 95 resulted in diminutionof agonist [3H]DPDPE binding but no change of antagonist[3H]naltrindol binding.

The non-specific, low-affinity interaction of monovalentcations with GPCR was ascribed to the high ionic strength(Seifert and Wenzel-Seifert 2002); however, the simple in-crease of ionic strength in aqueous medium can not explainour results because the ionic strength of all salts used in ourexperiments was the same. The role of negatively chargedions in affecting GPCR signaling should be also considered asan important “additional factor” because Cl−was present in allsalts used in our work (Seifert 2001; Schnell and Seifert2010).

The polar head-group region of lipid bilayer was consid-ered by us as the possible “site”where the low-affinity type ofsodium effect might take place (Jurkiewicz et al. 2012). Theinteraction of Na+ with membrane lipids could, subsequently,influence membrane proteins in an allosteric manner via lipidmolecules. Therefore, the polar membrane–water interface ofHEK293 cell membrane was studied by Laurdan generalizedpolarization (Laudran GP; Parasassi et al. 1991, 1994).

The value of Laurdan GPex was not significantlyaltered at 150 mM and 200 mM NaCl, LiCl, KCl, andCsCl; however, at higher concentrations, a decrease offluorescence intensity at 440 nm (blue end of Laurdanemission spectrum) and an increase of intensity at490 nm (red end of Laurdan emission spectrum) becamesignificant. At 800 mM concentration, which was used

in studies of unilamellar vesicles with a net-negativesurface charge (POPC containing 20 mol% of POPS;Jurkiewicz et al. 2012), monovalent cations caused ahighly significant decrease of Laurdan GPex when com-pared with the ion-free buffer: LiCl (p<0.0001), NaCl(p<0.0001, and KCl (p<0.0002). The effect of CsClwas not significant (Table 2). Expressed in quantitativeterms as an average of all GPex (λex) collected between320 and 400 nm at 20 °C, the following values of themean GPex (av) were obtained: 0.0995±0.0006 (Li+),0.0809±0.0009 (Na+), 0.0704±0.0007 (K+), 0.0651±0.0010 (Cs+), and 0.0657±0.0006 (ion-free). The effectof monovalent cations on Laurdan GPex (av) proceededin the order Li+>Na+>K+>Cs+≈ ion-free.

Virtually the same results were obtained by measurementsat 40 °C (Table 2). At this temperature, the values of LaurdanGP (av) in all media were lower than at 20 °C and reachednegative values because the intensity of fluorescence signal at490 nm was higher than at 440 nm. The increase of LaurdanGPex (av) declined again in the order Li

+>Na+>K+>Cs+≈ion-free. Thus, the magnitude of the effect of monovalent cationson polar head-group region of HEK293 membranes wasclearly related to average diameter (size) and charge densityof the given ion (see discussion for more details).

Studies of hydrophobic plasma membrane interiorby steady-state anisotropy of DPH

As the last experiment of our work dealing with δ-OR-Gi1α-HEK293 membranes, we have tested the effect of monovalentcations on the hydrophobic membrane interior and found nochange at all. Biophysical characteristics of the membraneinterior were determined by measuring steady-state fluores-cence anisotropy of hydrophobic membrane probe DPH(rDPH) as described by Brejchova et al. (2011). The resultsindicated no change of rDPH values by 100 mM, 200 mM, and800 mM NaCl, KCl, LiCl, and CsCl (Table 3).

Table 2 Average of Laurdan GPex (λex) values in δ-OR-Gi1α-HEK293 membranes

Solution GPex (av)

20 °C p Δ 40 °C p Δ

LiCl 0.0995±0.0006 <0.0001 0.0338 −0.1590±0.0012 <0.0001 0.0307

NaCl 0.0809±0.0009 <0.0001 0.0152 −0.1715±0.0012 <0.0001 0.0182

KCl 0.0704±0.0007 <0.0002 0.0047 −0.1825±0.0011 <0.0026 0.0072

CsCl 0.0651±0.0010 NS −0.0006 −0.1876±0.0014 NS 0.0021

Free 0.0657±0.0006 0 −0.1897±0.0011 0

The Laurdan GPex (λex) values collected between 320 and 400 nmwere merged, averaged, and expressed as GPex (av)±SEM. Statistical significance ofthe difference between GPex (λex) collected in ion-free buffer and media containing 800 mM LiCl, NaCl, KCl, and CsCl, respectively, was analyzed byunpaired Students t test using GraphPadPrizm4 software. NS, not-significant difference (p>0.05). Δ was calculated as the difference between GPex (av)values in media containing different ions and ion-free buffer (Free)

Naunyn-Schmiedeberg's Arch Pharmacol

Laurdan generalized polarization in POPC/POPS vesicles

To obtain information about the interaction of monovalentcations with a negatively charged lipid bilayer of definedchemical composition, we used a model system of unilamellarvesicles consisting of a mixture of negatively charged POPS(20 mol%) and zwitterionic POPC. Here, the scope of ourprevious study (Jurkiewicz et al. 2012) was extended to in-clude LiCl. The measurement of Laurdan GPex (av) wasperformed with the aim to obtain data comparable with thosecollected in the analysis of δ-OR-Gi1α-HEK293 membranes.

Small (SUV) or large (LUV) unilamellar vesicles com-posed of POPC/POPS were suspended in 800 mM or1,000 mM salt solutions and measured at 10 °C and 20 °C(Table 4A). These two salt concentrations and temperatureswere chosen to allow direct comparison between the previ-ously published data on lipid vesicles (1,000 mM salts at10 °C; Jurkiewicz et al. 2012) and δ-OR-Gi1α cell membranes(800 mM salts at 20 °C and 40 °C). The mean GPex (av)values were significantly increased by all salts when com-pared with ion-free medium. This increase proceeded in theorder LiCl>NaCl>KCl>CsCl>ion-free medium (Table 4A).The same order was found for both SUVat 10 °C and LUVat20 °C. The difference between the two sets of data was due tothe temperature difference as the POPC/POPS bilayer wasclearly more mobile at 20 °C than at 10 °C. The higher GPex(av) values reflect a more tightly packed lipid bilayer contain-ing less hydrated and less mobile lipid molecules at theglycerol level. Thus, the results obtained by measurementsof SUVat 10 °C and LUVat 20 °C were fully consistent withthe previously published solvent relaxation data (Jurkiewiczet al. 2012) and with “Hofmeister ordering” (Hofmeister1888) of different cations.

The strongest effect was found for lithium, which is likelyto have a qualitatively similar effect on the lipid bilayer assodium, which was shown to penetrate the bilayer and bind tolipid carbonyls by bridging them. Sodium also restricts thefreedom of motion of lipid head-groups at the glycerol leveland leads to bilayer dehydration (Jurkiewicz et al. 2012). Allinvestigated ions rigidified the POPC/POPS membrane. The

Table 3 Steady-state anisotropy of DPH fluorescence (rDPH) in δ-OR-Gi1α-HEK293 membranes

Ion-free 100 mM 200 mM 800 mM0.204±0.003 − − −

LiCl 0.205±0.004 0.209±0.004 0.211±0.004

NaCl 0.203±0.004 0.206±0.004 0.213±0.005

KCl 0.205±0,003 0.208±0.004 0.213±0.005

CsCl 0.208±0.004 0.206±0.004 0.214±0.004

(+)NMDG 0.207±0.004 0.211±0.004 0.216±0.005

The stock membrane suspension was diluted with 50 mM Tris–HCl,1 mM EDTA, pH 7.4 containing 100, 200, or 800 mM LiCl, NaCl,KCl, CsCl, and (+)-NMDG to 0.1 mg/ml; the 1 mM DPH in acetonwas added quickly under stirring (1 μM final concentration) and incubat-ed for 30 min at 25 °C. Steady-state anisotropy of DPH fluorescence wasmeasured at Ex 365/Em 425 nmwavelengths. Data represent the averageof three sets of measurements±SEM

Table 4 Average of Laurdan GPex values in POPC/POPS (A) and POPC/POPS/cholesterol (B) lipid vesicles

A. POPC/POPS lipid vesicles

Solution GPex (av)

10 °C p Δ 20 °C p ΔLiCl 0.2941±0.0010 <0.0001 0.0703 −0.0481±0.0019 <0.0001 0.1082

NaCl 0.2748±0.0013 <0.0001 0.0510 −0.0862±0.0019 <0.0001 0.0701

KCl 0.2566±0.0013 <0.0001 0.0328 −0.0992±0.0019 <0.0001 0.0571

CsCl 0.2470±0.0013 <0.0001 0.0232 −0.1293±0.0019 <0.0001 0.0270

Free 0.2238±0.0014 0 −0.1563±0.0022 0

B. POPC/POPS/cholesterol vesicles

20 °C p Δ 40 °C p ΔLiCl 0.3423±0.0003 <0.0001 0.0483 0.1194±0.0003 <0.0001 0.0877

NaCl 0.3265±0.0003 <0.0001 0.0325 0.1002±0.0003 <0.0001 0.0685

KCl 0.3236±0.0003 <0.0001 0.0296 0.0903±0.0003 <0.0001 0.0586

CsCl 0.3180±0.0003 <0.0001 0.0240 0.0902±0.0003 <0.0001 0.0585

Free 0.2940±0.0003 0 0.0317±0.0004 0

(A) Laurdan general polarization (Laurdan GP) was measured at 10ºC (LUV) and 20ºC (SUV) in liposomes composed of POPC and POPS in a 4:1,molar ratio. (B) Laurdan GP was measured at 20 °C and 40 °C in LUV composed of POPC (46 mol%)/POPS (20 mol%)/cholesterol (33 mol%).Numbers represent the average of Laurdan GPex (λex) values [GPex (av)] collected at excitation wavelengths (λex) 320–400 nm±SEM. in POPC/POPS orPOPC/POPS/cholesterol vesicles. Statistical significance of the difference between GPex (av) in ion-free buffer and media containing 800 mM LiCl,NaCl, KCl or CsCl, respectively, was analyzed by unpaired Students t test using GraphPadPrizm4 software.NS, non-significant difference (p>0.05). TheΔ represents the difference between GPex (av) values in the presence and absence of ions

Naunyn-Schmiedeberg's Arch Pharmacol

effectiveness of the monovalent ions was clearly related to thecharge density: lithium effect was the strongest, followed bysodium, potassium, and cesium (LiCl>NaCl>KCl>CsCl).

Laurdan generalized polarization in large unilamellarPOPC/POPS/cholesterol vesicles

Data obtained in the study of Laurdan GP in negativelycharged POPC/POPS vesicles were extended by studyingLUV composed of POPC/POPS and cholesterol (Table 4B).Cholesterol was included in the lipid mix with the aim tocreate a lipid bilayer more similar to natural HEK293 mem-branes containing this highly hydrophobic and electricallyneutral membrane constituent in high amounts (Brejchovaet al. 2011; Ostasov et al. 2013). Results obtained at bothtemperatures of measurement, 20ºC and 40ºC, were the sameas those obtained in POPC/POPS vesicles alone (Table 4A):the effect of monovalent ions decreased in the order LiCl>NaCl>KCl>CsCl>ion-free. Comparison with HEK293membranes indicated a much stronger effect for all ions (aboutthreefold).

It may be therefore concluded that the effect of high con-centrations of monovalent ions (>200 mM) on membranesprepared from HEK293 cells proceeds primarily as an inter-action with the surface, polar-head group region of lipidbilayer. The hydrophobic membrane interior does not partic-ipate in this effect (Table 3). The ion-induced change may beinterpreted as decreased hydration and decreased lipid mobil-ity at the membrane-water interface. The similarity of LaurdanGP data collected in HEK293 membranes (Table 2) andnegatively charged lipid vesicles (Table 4) suggests that thenegative surface charge is attracting cations which penetratedown to the lipid glycerol level in the polar head-group regionof the membrane. Cations are bridging the neighboring lipidsand replace water molecules. The effect is directly proportion-al to charge density of the un-hydrated form of these cationsand decreases in the order Li+>Na+>K+>Cs+≈ ion-freemedium.

Discussion

Comparison of the effect of sodium, potassium, lithium and (+)-NMDG on ligand binding to δ-OR-Gi1α (C351-I351) fusionprotein (inhibition of agonist and increase of antagonist bind-ing) indicated preferential sensitivity to sodium (Figs. 1 and2). Lithium, whose atomic radius is similar to that of sodium,was the second most effective ion in this respect. The detailedanalysis performed in a wide range of concentrations indicatedthat the overall, inhibitory effect of sodium on agonist bindingto δ-OR-Gi1α (C351-I351) is composed of two components,i.e., high- and low-affinity interaction sites.

Preferential sensitivity of agonist binding to sodium wasintensively documented in the past, but our data bring the firstevidence for the existence of low-affinity Na+ sites togetherwith high-affinity Na+ sites. The clear identification of lithiumas the second most effective ion had not been often noticedand, in support of our findings, the same order of specificity,Na+≫Li+>K+>choline+, was detected in NG108-15 cell lineby Blume et al. (1979).

Models of 3D structure of δ-, μ- and κ-opioid receptors(Pogozheva et al. 1998) revealed that each receptor structurehas an extensive network of interhelical hydrogen bonds. Theligand binding pocket interacts with the tyramine moiety ofalkaloids or Tyr1 of opioid peptides via conserved a.a. resi-dues in the core bottom of the pocket. Tyramine N+ and OH–

groups form ionic interactions or H-bonds with a conservedaspartate in 3TM and a conserved histidine in 6TM.Conserved Asp95 in trans-membrane helix II (2TM) of δ-OR is responsible for the lowering of agonist binding bysodium and interaction with G proteins (Chakrabarti et al.1997; Kong et al. 1993; Surratt et al. 1994). Within the 3Dstructure of δ-OR, Asp95 (2TM) participates in H-bond net-work with the polar residues of Asn131 (3TM), Ser135(3TM), Ser311 (7TM), and Asn314 (7TM; Lomize et al.1999). Binding of Na+ distorts this H-bond network.

As already mentioned in introduction, the most clear-cutdata dealing with the problem of water accessibility of ligandbinding site of OR are based on crystal structure of δ-OR andμ-OR with antagonists naltrindol and morphinan, respectively(Granier et al. 2012; Manglik et al. 2012; Filizola and Devi2012). Compared to the “buried” character of ligand bindingpocket in most other GPCRs, OR antagonists bind within alarge solvent-exposed pocket. The crystal structure of A2A

adenosine receptor (A2AAR) contains 177 structured watermolecules, 57 of which occupy the interior of the 7TM bundle(Liu et al. 2012). Interior water molecules form an almostcontinuous channel extending from the ligand binding site tothe site of G protein interaction, which is composed of threebulky water molecule clusters as well as several scatteredwater molecules. The channel has two narrow “bottlenecks”restricted by Trp2466.48 and Tyr2887.53 to slightly less than thediameter of one water molecule, 2.4 and 2.0 Å, respectively.Rearrangements of the receptor backbone and side chainsupon A2AAR-agonist binding (in the active state) disrupt thewater channel continuity and substantially reduce its volume.

The most recent study of the high-resolution (1.8 Å)crystal structure of antagonist (natrindol)-bound δ-OR re-vealed the fundamental role of sodium ion in mediatingallosteric control of receptor functional selectivity and con-stitutive activity (Fenalti et al. 2014). Sodium ions wereshown to stabilize a reduced agonist affinity state and mod-ulate signal transduction. Curiously, the Kd=7.9 mM ofhigh-affinity sites for Na+ determined in δ-OR-Gi1α (C351-I351) (Fig. 1) is very close to KB=13.3 mM, presented in

Naunyn-Schmiedeberg's Arch Pharmacol

Tab. 1 by Fenalti et al. (2014). Similarly to our study, theseauthors performed [3H]DADLE binding experiments in me-dia containing up to 400 mM NaCl; at 100 mM concentra-tion of chloride salts of different ions, the inhibition of[3H]DADLE binding to BRIL-δ-OR(ΔN/ΔC) was signifi-cant for sodium only, however, Li+ represented the secondmost effective cation.

When combining these literature data with the results pre-sented in this work, we suggest that the specific, high-affinitysites for sodium in δ-OR-Gi1α (C351-I351)-HEK293 mem-branes (Fig. 1a) are localized deep in the interior of the ligandbinding pocket and mediate an “inverse agonist-like” effect ofsodium (R*→R transition). The low-affinity Na+ sites, whichmediate the low-affinity type of inhibition of [3H]DADLEbinding, are represented by amino acids and a polar head-groups of tightly bound lipids in a water accessible area of thebinding pocket. The tightly bound lipids overlap with the lipidmolecules present in a water-accessible area of nearby “lipid-shell” and with the continuum of lipids in membrane lipidbilayer (Anderson and Jacobson 2002). Potassium and lithiumcompete with sodium for the low-affinity sites only.

Agonist-induced release of sodium ions from thespecific sites located in the interior of binding pocket(R→R* transition) proceeds as the first step in a con-formational change of receptor protein, followed in timeby the second phase, when released sodium is mixedwith potassium and other ions present in the “outerarea” of the binding pocket exposed to bulk-water/aque-ous space. This shift is associated with an onset ofcompetition of sodium with potassium and lithium forlow-affinity sites.

Preferential ability of lithium to support the potency/affinity of G protein response may be interpreted as anatypical feature of the receptor fusion protein distinctfrom the wild type receptor (Table 1 and Fig. 3).Interestingly, the same result was obtained for activationof Gi1α (C351-I351) plus endogenous G-proteins mea-sured in membranes prepared from PTX-untreated cells(Table 1B). The question to what extent this “specialfeature” of δ-OR-Gi1α (C351-I351) may reflect or mimicthe state of δ-OR-G protein coupling in the pathologicalsituation of a chronic exposure of brain to lithiumremains to be answered in the future (Young 2009).This type of analysis is difficult to perform as mem-branes isolated from rat brain exhibit a very smallstimulation of [35S]GTPγS binding or [32P]GTPase ac-tivity by δ-OR agonists. In δ-OR-Gi1α (C351-I351) cells,a significant decrease of the basal level of [35S]GTPγSbinding was detected at 1 mM (96 %) and 2 mM(92 %) LiCl (Table 5).

Biophysical analysis of the effect of sodium, potassi-um, lithium, and cesium on δ-OR-Gi1α (C351-I351)-HEK293 membranes by Laurdan GP (Table 2) indicatedonly the low-affinity type of interaction in the orderLi+ >Na+>K+>Cs+. The interaction of ions with

Table 5 Decrease of [35S]GTPγS binding by 0.5, 1, and 2 mM LiCl

Ion-free 0.5 mM 1 mM 2 mM

(−) DADLE 100±1.5 97±1.5NS 96±1.6* 92±1.9*

(+) DADLE 100±1.7 97±1.5NS 96±1.6* 95±1.5*

[35 S]GTPγS binding was measured in P2 membranes prepared fromPTX-untreated δ-OR-Gi1α-HEK293 cells in the absence or presence of10−5 M DADLE as described in methods. Binding assays were per-formed in ion-free, 0.5, 1, and 2 mM LiCl±10−5 M DADLE. Numbersrepresent the means of 3 binding assays±SEM performed in triplicate.Data were expressed as % of the basal level of binding in the absence ofions (100 %). Data were analyzed by one-way ANOVA followed byBonferroni’s Multiple Comparison Test (*p<0.05, NS, p>0.05)

pm

ol x

mg

-1

0.0

0.5

1.0

1.5 A

0.0

0.5

1.0

B

Free NaCl NaBr NaI Free NaCl NaBr NaI

Fig. 4 Decrease of [3H]DADLE (a) and [35S]GTPγS binding (b) byNaCl, NaBr, and NaI. [3H]DADLE (a) and [35S]GTPγS (b) bindingassays were performed in ion-free and 145 mM NaCl, NaBr, and NaIcontaining media. P2 membrane fraction isolated from PTX-untreatedcells was used. Numbers represent the means±SEM of 3 binding assaysperformed in triplicate. The same membrane preparation was used in allassays. Data were analyzed by one-way ANOVA followed by

Bonferroni’s Multiple Comparison Test: a [3H]DADLE binding: ion-freeversus NaCl (*p<0.05), NaCl versus NaBr (NS, p>0.05), NaBr versusNaI (**p<0.01). b [35S]GTPγS binding [(−)DADLE]: ion-free versusNaCl (***p<0.001), NaCl versus NaBr (*p<0.05), NaBr versus NaI(**p<0.01). b [35S]GTPγS binding [(+)DADLE]: ion-free versus NaCl(**p<0.01), NaCl versus NaBr (**p<0.01), and NaBr versus NaI(***p<0.001)

Naunyn-Schmiedeberg's Arch Pharmacol

negatively charged POPC/POPS and POPC/POPS/cho-lesterol vesicles decreased in the same order: Li+>Na+>K+>Cs+ (Table 4) and was interpreted as de-creased hydration and mobility of the polar head-groupregion of lipid bilayer. Interestingly, comparison be-tween HEK293 membranes and lipid vesicles may rep-resent a contribution to the field of biophysical studiesof specific ionic effects related to the early works ofFranz Hofmeister (Hofmeister 1888; Kunz et al. 2004).The so-called Hofmeister series of ions have been foundto be very similar for many different phenomena ob-served at complex macromolecular, polymeric and bio-logical interfaces. There is, however, a growing evi-dence for the complex origin of these effects (Faraday2013) and numerous experimental systems have beenidentified in which the Hofmeister series can be partlyor completely reversed (Parsons et al. 2010; Schwierzet al. 2010). Such reversed Hofmeister series have beenfound here by analysis of Laurdan GP in natural mem-branes isolated from HEK293 cells (Li+>Na+>K+>Cs+)and in model lipid bilayers composed of negativelycharged POPC/POPS or POPC/POPS/cholesterol (Li+>Na+>K+>Cs+).

In this context, the role of counter anions has to bealso considered. The iodide anions (I−), being morehydrophobic than Cl− and Br−, were found to penetratemore deeply into the membrane bilayer (Jurkiewiczet al. 2011); simultaneously, iodide salts of monovalentcations were more efficient than chloride salts whenmodulating agonist binding and G protein coupling ofβ2-adrenergic, chemokine CXCR4 and histamine H3receptors (Seifert 2001; Kleemann et al. 2008; Schnelland Seifert 2010). Our data confirmed these results. Theinhibition of [3H]DADLE binding increased in the orderNaCl<NaBr<NaI (Fig. 4a). The same order was foundfor inhibition of the basal and DADLE-stimulated[35S]GTPγS binding (Fig. 4b).

Conclusions

The importance of our work is based on (a) distinction be-tween high- and low-affinity sites for Na+ which has not beenpresented in the current literature and (b) simultaneous com-parison of the effect of sodium, potassium and lithium on theagonist and antagonist binding, G protein coupling, polarhead-group region and hydrophobic interior of HEK293 plas-ma membrane containing the δ-OR-Gi1α (C351-I351) hybridprotein. The low-affinity type of interaction of monovalentcations with δ-OR-Gi1α (C351-I351)-HEK293 membrane isinterpreted as binding to a water-accessible part of the ligandbinding pocket and polar head-groups of lipid molecules.

Acknowledgments This work was supported by the Grant Agency ofCzech Republic (P207/12/0919 and P304/12/G069) and by Academy ofSciences of the Czech Republic (AV0Z50110509 and RVO:67985823).

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