The β1B Subunit Regulates the Activity of LVA Calcium Channels: Evidences for a Physical...
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change in the pore size due to the intrinsic heterogeneity in the fluorescently labeled MscL pentamers where various stoichiometry of the donors and accep- tors are present. Furthermore, we have found that sticking the channel to a sur- face alters the function of them. To get around these two problems, we performed single-molecule FRET experiments using an Alternating Laser EX- citation (ALEX) apparatus. By using alternating lasers of wavelengths 488 nm and 561 nm to excite directly the donors (Alexa488) and acceptors (Alexa568) present in single diffusing MscL molecules which are incorporated in 50 nm liposomes, both the distance-based FRET efficiency E and stoichiometry- based ratio S could be measured. Our single-molecule experiments show that the addition of an asymmetric lipid (LPC) to the liposomes opens the channels, consistent with the results from ensemble measurements. Voltage-gated Ca Channels 623-Pos Board B409 A Short Basic Segment within a Non-Conserved Region of the Beta- Subunit Modulates the Rate of Inactivation of the R-Type Calcium Channel Miranda-Laferte Erick 1 , Silke Schmidt 1 , Alan Neely 2 , Patricia Hidalgo 1 . 1 Institut fu ¨r Neurophysiologie; MHH, Hannover, Germany, 2 CINV-U, de Valparaiso, Valparaiso, Chile. Besides opening and closing, high-voltage activated (HVA) calcium channels transit to an inactivated state from which they do not re-open unless the plasma membrane is repolarized. This process is critical for the temporal regulation of intracellular calcium signaling. Ca V 2.3, a particular class of HVA channels supporting R-type calcium currents, inactivates fully in few hundred millisec- onds when expressed alone but when co-expressed with the palmitoylated form of the regulatory b-subunit (Ca V b 2a ), this process is slowed several fold and is incomplete. The widely accepted view is that membrane-anchoring of the Ca V b 2a immobilizes the channel inactivation machinery. Some evidence sug- gests that an additional structural determinant may be coded in the linker pep- tide that joins the two highly conserved domains composing Ca V b but whether these determinants target the subunit to the membrane has not been shown. Here we indentify a short positively charged segment lying at the boundary of the guanylate kinase domain of Ca V b 2a that slows down channel inactivation without relocating the protein to the plasma membrane. Deletion analysis dem- onstrates that while neither the N-terminal nor the C-terminal variable region of the protein affects the regulatory effect of the basic segment the presence of the remaining residues within the linker does. These results demonstrate that mem- brane anchoring is not the only factor modulating inactivation rate Ca V 2.3 cal- cium channels and suggest the presence of intralinker interactions or posttranslational modifications that counteract the effect of this segment. 624-Pos Board B410 The b1B Subunit Regulates the Activity of LVA Calcium Channels: Evidences for a Physical Interaction Rogelio Arteaga-Tlecuitl, Clara E. Dı ´az-Vela ´squez, Manuel Rivera, Juan C. Gomora. Instituto de Fisiologı ´a Celular, UNAM, Mexico City, Mexico. Voltage-gated Ca2þ (CaV) channels are classified as Low Voltage- (LVA; or CaV3 channels) and High Voltage- (HVA) Activated channels. Both are formed by a main a1 subunit, which forms the conduction pore and senses the changes in membrane potential. It consists of four domains (I-IV), which are similar to one another and contain six transmembrane segments (S1-S6). Auxiliary subunits (b, a2d y g) have been described only for HVA channels; they regulate the main subunit expression level, voltage dependence and cur- rent kinetics. The loop between domain I and II of HVA a1 subunits contains a conserved sequence known as AID, where a1 and b subunits interaction take place. Despite such domain is not present in LVA a1 subunits, it has been shown that cells expressing heterologously LVA channels significantly in- crease its current density in the presence of b subunits. Recently it has been sug- gested a weak interaction between peptides of the I-II loop of CaV3.3 channels and the core of b1 subunit. Nevertheless this interaction has not been shown at the whole protein level. The main goal of this work was to investigate whether b subunits modulate LVA Ca2þ channels and to determine if these two pro- teins interact physically. By using the whole cell patch clamp technique, we found that b1b subunit increases the current density (63, 72 and 45 %) of HEK-293 cells expressing CaV3.1, CaV3.2 and CaV3.3, respectively. Then, with fusion fluorescent proteins and confocal microscopy, we detected changes in b1b cellular distribution in the presence of the CaV3.3 channel. Finally, by western blot we identified the protein complex formed by Cav3.3 and b1b. Al- together, these data suggested that HVA b subunits modulate LVA Ca2þ chan- nels probably by a physical interaction. CONACYT-Mexico J50250Q. 625-Pos Board B411 Differential Requirement of GTP-Binding for Rem and Rad Inhibition of Ca V 1.2 Channels in Heart Donald Chang, Henry M. Colecraft. Columbia University, New York, NY, USA. Ca 2þ influx through L-type (Ca V 1.2) calcium channels in heart controls: action potential duration; muscle contraction; and gene expression. Rad/Rem/Rem2/ Gem/Kir (RGK) GTPases are Ras-like monomeric G-proteins that bind auxiliary Ca V channel beta subunits and potently inhibit Ca V 1/Ca V 2 channels. RGK pro- teins are expressed in heart, where their expression levels changes in disease. Whether GTP-binding is important for RGK-mediated inhibition of Ca 2þ cur- rent (I Ca,L ) in heart is controversial. Here, we investigated whether GTP binding to Rem and Rad is important for their ability to inhibit Ca V 1.2 channels. Both wild-type (wt) Rem and Rad dramatically inhibited recombinant Ca V 1.2 chan- nels reconstituted in HEK 293 cells. Putative GTP-binding-deficient mutants (RemT94N and RadS105N) similarly inhibited recombinant I Ca,L . In heart cells (Figure), over-expressing wt Rem or Rad (black lines) potently inhibited endog- enous I Ca,L (gray lines). Surprisingly, RemT94N (solid triangles) was function- ally inert in heart, whereas RadS105N (solid squares) in- hibited I Ca,L to the same extent as wt Rad. The results con- tradict reports that RadS105N acts as a dominant negative in heart, and reveals a novel intrinsic dif- ference between the two RGK proteins. 626-Pos Board B412 Rare Missense Mutations in the Calcium Channel b2 Subunit Gene of Autistic Patients Reduce Time-Dependent Inactivation of Ca V 1.2 Alexandra F. Breitenkamp 1 , Judith Sinzig 2 , Peter Nu ¨rnberg 3 , Stefan Herzig 1 . 1 Department of Pharmacology, University of Cologne, Cologne, Germany, 2 Department of Child & Adolescent Psychiatry and Psychotherapy, University of Cologne, Cologne, Germany, 3 Cologne Center of Genomics, University of Cologne, Cologne, Germany. Loss of function of voltage-dependent L-type calcium channels (VDCC) in pa- tients with Timothy syndrome results in a multiorgan dysfunction including le- thal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. This single gene disorder serves as a model disease for autism spectrum disease (ASD), giving insights in a possible pathophysiology. A point mutation in the pore-forming calcium channel subunit Ca V 1.2 gene is involved in the de- velopment of the Timothy syndrome and leads to incomplete inactivation of the L-type calcium currents (Splawski et al., Cell 2004;119:19-31). Functionally similar biophysical effects can be induced by the influence of auxiliary VDCC b subunits (Herzig et al., FASEB J. 2007;21:1527-38; Jangsangthong et al., Pflugers Arch. 2010;459:399-411). Supported by findings in a meta-analysis of linkage data of ASD patients (Trika- linos et al., Mol Psychiatry. 2006;11:29-36), we are investigating a function-based candidate gene hypothesis linking the b2 subunit gene (CACNB2) with ASD. We performed a case control study sequencing all exons and flanking introns of CACNB2 in 155 patients. We found three rare missense mutations in ASD pa- tients, but not in 259 unaffected controls. All three occur at highly conserved po- sitions and might alter protein function; additionally one mutation probably introduces a new phosphorylation site. Until now, we characterized two of these mutations and a phosphorylation-mimicking mutant in electrophysiological stud- ies. All variants show a decelerated and incomplete time-dependent inactivation of the co-transfected Ca V 1.2 subunit. So far two variants exhibit a significant in- creased slope factor of voltage-dependent steady-state inactivation. We here present mutations in the b2 subunit gene of ASD patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic Tim- othy syndrome mutations of Ca V 1.2. b2 subunit mutations may influence neu- ronal function or development in some ASD patients. 124a Sunday, February 26, 2012
Transcript of The β1B Subunit Regulates the Activity of LVA Calcium Channels: Evidences for a Physical...
124a Sunday, February 26, 2012
change in the pore size due to the intrinsic heterogeneity in the fluorescentlylabeled MscL pentamers where various stoichiometry of the donors and accep-tors are present. Furthermore, we have found that sticking the channel to a sur-face alters the function of them. To get around these two problems, weperformed single-molecule FRET experiments using an Alternating Laser EX-citation (ALEX) apparatus. By using alternating lasers of wavelengths 488 nmand 561 nm to excite directly the donors (Alexa488) and acceptors (Alexa568)present in single diffusing MscL molecules which are incorporated in 50 nmliposomes, both the distance-based FRET efficiency E and stoichiometry-based ratio S could be measured. Our single-molecule experiments show thatthe addition of an asymmetric lipid (LPC) to the liposomes opens the channels,consistent with the results from ensemble measurements.
Voltage-gated Ca Channels
623-Pos Board B409A Short Basic Segment within a Non-Conserved Region of the Beta-Subunit Modulates the Rate of Inactivation of the R-Type CalciumChannelMiranda-Laferte Erick1, Silke Schmidt1, Alan Neely2, Patricia Hidalgo1.1Institut fur Neurophysiologie; MHH, Hannover, Germany,2CINV-U, de Valparaiso, Valparaiso, Chile.Besides opening and closing, high-voltage activated (HVA) calcium channelstransit to an inactivated state from which they do not re-open unless the plasmamembrane is repolarized. This process is critical for the temporal regulation ofintracellular calcium signaling. CaV2.3, a particular class of HVA channelssupporting R-type calcium currents, inactivates fully in few hundred millisec-onds when expressed alone but when co-expressed with the palmitoylated formof the regulatory b-subunit (CaVb2a), this process is slowed several fold and isincomplete. The widely accepted view is that membrane-anchoring of theCaVb2a immobilizes the channel inactivation machinery. Some evidence sug-gests that an additional structural determinant may be coded in the linker pep-tide that joins the two highly conserved domains composing CaVb but whetherthese determinants target the subunit to the membrane has not been shown.Here we indentify a short positively charged segment lying at the boundaryof the guanylate kinase domain of CaVb2a that slows down channel inactivationwithout relocating the protein to the plasma membrane. Deletion analysis dem-onstrates that while neither the N-terminal nor the C-terminal variable region ofthe protein affects the regulatory effect of the basic segment the presence of theremaining residues within the linker does. These results demonstrate that mem-brane anchoring is not the only factor modulating inactivation rate CaV2.3 cal-cium channels and suggest the presence of intralinker interactions orposttranslational modifications that counteract the effect of this segment.
624-Pos Board B410The b1B Subunit Regulates the Activity of LVA Calcium Channels:Evidences for a Physical InteractionRogelio Arteaga-Tlecuitl, Clara E. Dıaz-Velasquez, Manuel Rivera,Juan C. Gomora.Instituto de Fisiologıa Celular, UNAM, Mexico City, Mexico.Voltage-gated Ca2þ (CaV) channels are classified as Low Voltage- (LVA; orCaV3 channels) and High Voltage- (HVA) Activated channels. Both areformed by a main a1 subunit, which forms the conduction pore and sensesthe changes in membrane potential. It consists of four domains (I-IV), whichare similar to one another and contain six transmembrane segments (S1-S6).Auxiliary subunits (b, a2d y g) have been described only for HVA channels;they regulate the main subunit expression level, voltage dependence and cur-rent kinetics. The loop between domain I and II of HVA a1 subunits containsa conserved sequence known as AID, where a1 and b subunits interaction takeplace. Despite such domain is not present in LVA a1 subunits, it has beenshown that cells expressing heterologously LVA channels significantly in-crease its current density in the presence of b subunits. Recently it has been sug-gested a weak interaction between peptides of the I-II loop of CaV3.3 channelsand the core of b1 subunit. Nevertheless this interaction has not been shown atthe whole protein level. The main goal of this work was to investigate whetherb subunits modulate LVA Ca2þ channels and to determine if these two pro-teins interact physically. By using the whole cell patch clamp technique, wefound that b1b subunit increases the current density (63, 72 and 45 %) ofHEK-293 cells expressing CaV3.1, CaV3.2 and CaV3.3, respectively. Then,with fusion fluorescent proteins and confocal microscopy, we detected changesin b1b cellular distribution in the presence of the CaV3.3 channel. Finally, bywestern blot we identified the protein complex formed by Cav3.3 and b1b. Al-
together, these data suggested that HVA b subunits modulate LVA Ca2þ chan-nels probably by a physical interaction.CONACYT-Mexico J50250Q.
625-Pos Board B411Differential Requirement of GTP-Binding for Rem and Rad Inhibition ofCaV1.2 Channels in HeartDonald Chang, Henry M. Colecraft.Columbia University, New York, NY, USA.Ca2þ influx through L-type (CaV1.2) calcium channels in heart controls: actionpotential duration; muscle contraction; and gene expression. Rad/Rem/Rem2/Gem/Kir (RGK)GTPases areRas-likemonomericG-proteins that bind auxiliaryCaV channel beta subunits and potently inhibit CaV1/CaV2 channels. RGK pro-teins are expressed in heart, where their expression levels changes in disease.Whether GTP-binding is important for RGK-mediated inhibition of Ca2þ cur-rent (ICa,L) in heart is controversial. Here, we investigated whether GTP bindingto Rem and Rad is important for their ability to inhibit CaV1.2 channels. Bothwild-type (wt) Rem and Rad dramatically inhibited recombinant CaV1.2 chan-nels reconstituted in HEK 293 cells. Putative GTP-binding-deficient mutants(RemT94N and RadS105N) similarly inhibited recombinant ICa,L. In heart cells(Figure), over-expressing wt Rem or Rad (black lines) potently inhibited endog-enous ICa,L (gray lines). Surprisingly, RemT94N (solid triangles) was function-
ally inert in heart,whereas RadS105N(solid squares) in-hibited ICa,L to thesame extent as wtRad. The results con-tradict reports thatRadS105N acts asa dominant negativein heart, and revealsa novel intrinsic dif-ference between thetwo RGK proteins.626-Pos Board B412Rare Missense Mutations in the Calcium Channel b2 Subunit Gene ofAutistic Patients Reduce Time-Dependent Inactivation of CaV1.2Alexandra F. Breitenkamp1, Judith Sinzig2, Peter Nurnberg3,Stefan Herzig1.1Department of Pharmacology, University of Cologne, Cologne, Germany,2Department of Child & Adolescent Psychiatry and Psychotherapy,University of Cologne, Cologne, Germany, 3Cologne Center of Genomics,University of Cologne, Cologne, Germany.Loss of function of voltage-dependent L-type calcium channels (VDCC) in pa-tients with Timothy syndrome results in a multiorgan dysfunction including le-thal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia andautism. This single gene disorder serves as a model disease for autism spectrumdisease (ASD), giving insights in a possible pathophysiology. A point mutationin the pore-forming calcium channel subunit CaV1.2 gene is involved in the de-velopment of the Timothy syndrome and leads to incomplete inactivation of theL-type calcium currents (Splawski et al., Cell 2004;119:19-31). Functionallysimilar biophysical effects can be induced by the influence of auxiliaryVDCC b subunits (Herzig et al., FASEB J. 2007;21:1527-38; Jangsangthonget al., Pflugers Arch. 2010;459:399-411).Supported by findings in a meta-analysis of linkage data of ASD patients (Trika-linos et al.,Mol Psychiatry. 2006;11:29-36),weare investigatinga function-basedcandidate gene hypothesis linking the b2 subunit gene (CACNB2)withASD.Weperformed a case control study sequencing all exons and flanking introns ofCACNB2 in 155 patients. We found three rare missense mutations in ASD pa-tients, but not in 259 unaffected controls. All three occur at highly conserved po-sitions and might alter protein function; additionally one mutation probablyintroduces a new phosphorylation site. Until now, we characterized two of thesemutations and a phosphorylation-mimickingmutant in electrophysiological stud-ies. All variants show a decelerated and incomplete time-dependent inactivationof the co-transfected CaV1.2 subunit. So far two variants exhibit a significant in-creased slope factor of voltage-dependent steady-state inactivation.We here present mutations in the b2 subunit gene of ASD patients that result ina retardation of inactivation behavior, thus phenocopying the monogenic Tim-othy syndrome mutations of CaV1.2. b2 subunit mutations may influence neu-ronal function or development in some ASD patients.
