Anion-Sensitive Regions of L-Type CaV1.2 Calcium Channels Expressed in HEK293 Cells 英文参考文献.docVIP

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Anion-Sensitive Regions of L-Type CaV1.2 Calcium Channels Expressed in HEK293 Cells 英文参考文献.doc

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Anion-Sensitive Regions of L-Type CaV1.2 Calcium Channels Expressed in HEK293 Cells 英文参考文献

Anion-SensitiveRegionsofL-TypeCaV1.2Calcium ChannelsExpressedinHEK293Cells NorbertBabai1,NatalyKanevsky2,NathanDascal2,GeorgeJ.Rozanski3,DhirendraP.Singh1 ,Nigar Fatma1,WallaceB.Thoreson1* 1DepartmentofOphthalmologyVisualScience,UniversityofNebraskaMedicalCenter,Omaha,Nebraska,UnitedStatesofAmerica,2DepartmentofPhysiologyand Pharmacology,SacklerSchoolofMedicine,TelAvivUniversity,RamatAviv,Israel,3DepartmentofCellularIntegrativePhysiology,UniversityofNebraskaMedicalCenter, Omaha,Nebraska,UnitedStatesofAmerica Abstract L-typecalciumcurrents(ICa)areinfluencedbychangesinextracellularchloride,butsitesofanioneffectshavenotbeen identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellularchloridewithgluconateorperchlorate.Variance-meananalysisofICa andcell-attachedpatchsinglechannel recordingsindicatethatgluconate-inducedinhibitionisduetointracellularanioneffectsonCa2+channelopenprobability, notconductance.InhibitionofCaV1.2currentsproducedbyreplacingchloridewithgluconatewasreducedfrom,75%– 80%to,50%byomittingbsubunitsbutunaffectedbyomittinga dsubunits.Similarly,gluconateinhibitionwasreduced 2 to ,50% by deleting an a1 subunit N-terminal region of 15 residues critical for b subunit interactions regulating open probability.Omittingbsubunitswiththismutanta1subunitdidnotfurtherdiminishinhibition.Gluconateinhibitionwas unchangedwithexpressionofdifferentbsubunits.TruncatingtheCterminusatAA1665reducedgluconateinhibitionfrom ,75%–80% to ,50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacementwithglutaminesreducedgluconateinhibitionto,60%indicatingtheseresiduesareparticularlyimportantfor anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to ,25% consistentwithadditiveinteractionsbetweenthetwotailregions.Ourresultssuggestthatmodestchangesinintracellular anionconcentrationcanproducesignificanteffect