Ion-channel-introduction-and-Na(2016)(ppt文档).ppt

ION CHANNELS Theories and Research Methods（A Course for PostgraduatesA Course for Postgraduates）Jian-Guo Chen,professorJian-Guo Chen,professorDepartment of PharmacologyDepartment of PharmacologyTongji Medical CollegeTongji Medical CollegeHUSTHUSTChapter 1,IntroductionChapter 1,IntroductionPart I:Concept of ion channelPart I:Concept of ion channel IonchannelsareporesconsistofproteinmoleculesinthecellmembraneIonchannelsaretheexcitablecomponentsofexcitablecellsIonchannelsmediatetheelectro-signalsonneurons,musclesandsynapsesIonchannelscodethesignalsandresponsesinnervoussystembyopening,closingandsynergyingAbout3millionyearsago,thelipidbilayermembranedeveloped,thisdual-moleculebarrierseparatesthelivingcellandtheenvironment:Advantages:thecompositionofcellsurvivalcanbereservedDisadvantages:ionizedsubstratesarenotaccessible,anditisdifficulttoexcludeionizedwasteproductionCreate a new transport mechanism became more urgent:Oneapproach:toestablishthemembranepores:largeenoughtoallowsmallmoleculemetabolitestopass,butalsosmallenoughtokeepmacromolecularmaterials-G-bacteriaandmitochondrialoutermembranearebuiltaccordingtothisdesign.Anotherapproach:thedesignismoresophisticated,morediverse,moreselectivetoaccomplishdifferenttasks-mostofthecytoplasmicmembraneissodesignedHowdothesemembranetransportdeviceswork?Before1980,ourunderstandingoftheirphysiologicalsubstance derived from the flow detection(based onmolecularselectivity,saturationconcentration,combinedwiththenumberofmolecules,etc.):Traditionally,theywerecategorizedas:CarrierandPoreCarrier:Atransporter,likeaboat,carryingwithsmallmolecules by its specific binding site across themembrane.Pore:A narrow,water-filled tunnel,allows a smallnumberofionsandsmallmoleculesthatfittheirporepermeabilitypassthroughthem.As the protein purification techniques and thedevelopmentofmolecularbiologytechniques,thecarrieroftheferryhypothesiswasrejected,asmanyofthepurifiedorclonedcarriersareproteinmolecules-theyaretoolarge,itisimpossibletopenetrate or rotate at the desired rates;theiramino acid residues form a linear foldedtransmembranesegment.New hypothesis:the membrane molecules isrelativelyfixed,theslightmovementislimitedwithinthe protein,binding sites on the membrane areexposed to the inside and outside.Such as K+-Na+pump,Ca2+pump,Na+-Ca2+exchangerThisnewinferenceneedstobefurtherverified.A crystal structure of the first carrier thatidentifiedin2000supportthisnewpointofview.Withcontrastdestinytoferryhypothesis,anothertransportmechanism-aqueouspore,wasconfirmed:During 1965-1980 years,the interaction betweenresearches on excitable membranes and on poremodels has greatly accelerated and deepened ourunderstandingofthemechanismsofthistransporter.In 80s,a landmark technological advance-patch-clamptechnique(NeherandSakmann)wasinvented,leadingtoanimportantdiscovery:ionsthroughthemembraneatarategreaterthan106/S,suchahighrate can be only competent by channel,but notothermechanisms.Patch-clampTechniquesEquipment:invertedmicroscope,micromanipulator,drug delivery systems,anti-vibrateworktable,etc.Patch-clamp amplifier,data acquisition devices,computers,acquisitionandanalysissoftware Electrodepuller,polishinginstrumentIonchannelsTheyaretransmembraneglycoproteins,theycanform hydrophilic pores,allowing charged ionspassthroughthecellmembrane.They are the basic excitable units in the cellmembranes of nervous,muscular,glandulartissues,theycanproduceandconductingelectricalsignals,mediatingimportantphysiologicalfunctions.PartII,thefunctionsofIonChannelsPhysiologistshavelongknownthationsplayakeyroleinexcitability.1881-1887,SidneyRingerdiscoveredthatthefrogheart perfusion solution must contain Na+,K+,Ca2+,and to a certain mixing ratio in order tomaintainthefrogheartbeatingforalongtime1888,WaltherNernstproposedtheviewpointthatthe potential originated from the penetration oftheelectrolytesolutionraisedalotofhypothesesaboutsourcesofbio-electricphenomena,suchasH+hypothesis1902,1912,Julius Bernstein rightlyproposed the following hypothesis:atrestingstatus,theexcitablecellmembraneonlypermitpermeabilitytoK+,Duringexcitement,themembranearepermeabletootherions.-This is the basis of modernmembranehypothesis.Inorganicionsmainlyrelatedwithmembraneexcitabilityare:Na+、K+、Ca2+、H+、Mg2+、Cl-、HCO3-、HPO42-Neuromuscular excitability and electricalsignals are mediated by the movement ofionsthroughcellmembrane.Na+,K+,Ca2+,Cl-mediate the most ofexcitatoryresponsesTheresponsesofChannelsareexpressedasOpeningandClosing,i.e:GatingAnopenedchannelhasselectivepermeability,it means that during channel opening,itonly allows certain ions flow along theelectrochemicalgradientsThispassivediffusionhastwocharacteristics:Rateishigh:106/secNoenergyWhichdistinguishesitfromothertransportmechanism.The open and close for an ion chnnelThe equivalent circuit of MembranePartIII,Thenomenclature In the 50s,ion channel is mainly based onmembranepermeability(channelkinetics,pharmacology,ionexchange,etc)toname.SuchasthatHodgkinandHuxleyinthesquidgiantaxonfoundontheNa+,K+currentsandsubsequentlyappropriatelynamedNa+,K+channels.Inthe80s,followingtherapiddevelopmentof molecular biology,a large number ofchannels were cloned,the correspondinggeneswerealsofound.This made the types and number of thechannels increased several times,such asthe types and subtypes of Na+,K+,Ca2+channelsaremorethan100.The former nomenclature could not meetthedemandofthenewdevelopments.Inaddition,thetraditionalnomenclatureaccordingtothepermeabilitycouldnotresolvethefollowingissues:Ifwedonotknowwhatkindofion,howtoname?Ifwedonotknowwhatisthemajorion,howtoname?If multiple channels using the same ion,how toname?ChannelNamingvaried:MolluscsgangliachannelA,B,C(Adam1980)CardiacPurkinjefiberschannelqr,si,x1(McAllister1975)Accordingtothenameofinhibitor:amiloride-sensitiveNa+channelsAccordingtonameofneurotransmitters:glycinechannelAccordingtothemutation:ShakerchannelAccordingtothedisease:CFTR(bladderfibrosistransmembraneregulator)Accordingtotheorderofthefindinglaboratory:GIRKSystemnomenclature:basedontherelationshipbetweenthechannelstructuresequenceandtheevolution.This strategy was firstly applied in thenomenclature of voltage-gated potassiumchannelsinmammalian:Kv1.1,Kv1.2.8.1(Chandy1991)Usingthismethods,theInternationalUnionof Pharmacology(IUPHAR)has namedother channels,such as calcium channelsandsodiumchannels.Channel family:There are many similarfunctionalcharacteristicsbetweendifferentchannels,such as voltage-gated Na+,K+,Ca2+channel,Acetylcholine,glycine andGABA have a lot of similarities betweenthem,According to the evolution of the channelhomology,theycanbedividedintodifferentsuperfamilyandfamily.PartIV,TwoEquationsOhmslaw：I=gEorE=IRNernstequation：R：Gasconstant，T：Absolutetemperature，F：FaradayconstantPartV,Ionchannelproperties(1)Selectivity:ionchannelmostimportantand the most significant feature is its ionselectivity.Selectivityisdeterminedby:Therelativesizeoftheminimumdiameterofionchanneltothesizeoftheion.The charged hydrophilic group in thechannelandthenatureofthecharge.Principle:Iso-electricattractionTwo cases in the channels with the samecharge:When channel contains weak polar sites:selectivityisrelevanttotheiondehydration(large ions are easy to dehydrationso iseasytopass)When channel contains strong polar sites:smallionsaremoreeasytopass（2）GatingIonchannelsareusuallyclosed,theywillopenonlywhenactivated.Basedontheirregulatorymechanisms,theycanbedividedinto:ligand-gatedionchannelvoltage-gatedionchannelThegatingofionchannelcontains3statuses:Voltage-gated channelLigand-gated channelChapterChapter 2,Sodium Channel 2,Sodium ChannelPart I.PrefaceNa+channels are widely expressed in the excitable cell membrane.It mediates the major current component of the rising phase of the action potential in excitable membrane.PartII.TheMolecularStructureofNa+ChannelThefirstionchannelgenecloneisobtainedfromthesodiumchannelofelectriceel.The cloned sodium channels from otheranimals possess the basic structure of theelectriceelsodiumchannel.Sodium channel is an oligomer,consist ofabout2000aminoacidresidues.Containing subunit(260KD),1 subunit(36-38KD),2subunit(33KD).Thesubunits,activationandinactivationofNa+channel-subunit has four homologous domains,eachdomain contains 300-400 amino acid residues,forming six transmembrane segments(S1-S6),inwhichtheaminoacidstakeshapeofhelixregion.S4isavoltagesensitiveareas,inwhichpositivelycharged residues,such as arginine or lysine,arerich,itfeelthechangeinmembranepotential.ThareareahairpinstructurebetweentheS5-S6,formingahydrophilicporeselectivetoions.Itiscalledchannelregion(poreregion),Itisalsothetargetofdrugsandtoxins.C-terminalandN-terminalareontheintracellularside.1subunitcontains213aminoacids,forminganhelix.C-terminal is on the intracellular side,N-terminalisontheextracellularside.1subunitchannelisinvolvedintheactivationandinactivationprocesses.Thefunctionof2subunitisunclear.Thefunctionof2subunitisunclear.If a Na+channel only contains an subunit,thechannelshowsaslowinactivation,When co-expressed with the 1 subunit,channelinactivationratecanbeincreasedbyfive-fold,thecurrentamplitudecanbeincreasedby2.5-foldPartIII.ThesystemicclassificationandnomenclatureofSodiumchannelI.e.I.e.I.e.I.e.Nav1.4ismainlyexpressedinskeletalmuscle;Nav1.4ismainlyexpressedinskeletalmuscle;Nav1.5primarilyinthemyocardiumNav1.5primarilyinthemyocardium.Theformerclassificationandnomenclature:Accordingtothedifferent subunits,itwasclassifiedas:I、II、III、1、h1、NaGetal.Part IV,The features of sodium channel currents1)ThedistributionsofNa+channelsindifferentorgansortissuesaredifferent,However,thedifferencebetweenNa+channelsisrelativesmallerthanthatbetweenK+,Ca2+channelsAccording to the voltage-dependent and the sensitivity toTTX,Na+channelscanbedividedinto:INa:voltage-gated Na+channels(TTX-sensitive Na+channels,fastNa+channels):APdepolarizationphaseINa,B or INa,S:Non-voltage-gated Na+channels(TTX-insensitive Na+channels and slow Na+channels,backgroundNa+currents):sinusnode,ithelpstheformationofthepacemakercurrentinthelateofphase4(If).(2)、ThefeatureINaWhenthepotentialisclampedat-70mv，Inaisaninwardcurrent，Voltagedependent，Thethresholdforchannelopening:-60-50mv,Theactivationandinactivationarerelativelyfastact=0.10.4ms,inact12ms;Mostofthechannelswillinactivatewithin45ms.(3)TheI-VcurveofINa：Activation:-60-50mvPeakamplitude:-25-30mvReversalpotential:+50mv（dependontheoncentrationbetweenthetwosides,indicatingtheEquilibriumpotentialof NaNa+）（4）TheinactivationandrecoverycurveofINaBefore the test pulse,the channels was given a long pulse(PrePulse),then test the non-inactivated current,and thennormalizedit.Paired-pulsetestTwo pulses at samepotential are given atdifferenttimeintervals,thenobservetherelationshipbetween time intervalandrecoveryI(2)/I(1)isplottedtointervals(5).ThefeaturesofINathesingleionchannelcurrentThe open of singlechannelisrandomizedThe mean open time:1ms，Conductance:7.820pSVh=-80mvVt=-40mvUnitarycurrent=2.2pAPart V,INa Blockers or ToxinsINainneuronsismoresensitivetoTTXthaninmyocardiumTheRecordingofINaTo test Na+currents,the pipette solutionshouldcontainCs+(Cesium)insteadofK+toblockK+current,theextracellularsolutionshould contain Ni2+(Nickel,50M)toblockICa-T,plusCd2+(Cadmium,100M)toblockICa-L.Beforetheendoftheexperiment,itshouldbeconfirmedwithTTX.50