(7.1)-拓展知识（1）离子通道与膜片钳技术2003c

TheNobelPrizeinChemistry2003wasawarded"fordiscoveriesconcerningchannelsincellmembranes"jointlywithonehalftoPeterAgre"forthediscoveryofwaterchannels"andwithonehalftoRoderickMacKinnon"forstructuralandmechanisticstudiesofionchannels."MolecularchannelsletusenterthechemistryofthecellWehumanbeingsconsisttoabout70%ofsaltwater.Thisyear’sNobelPrizeinChemistryrewardstwoscientistswhosediscoverieshaveclarifiedhowsalts(ions)andwateraretransportedoutofandintothecellsofthebody.Thediscoverieshaveaffordedusafundamentalmolecularunderstandingofhow,forexample,thekidneysrecoverwaterfromprimaryurineandhowtheelectricalsignalsinournervecellsaregeneratedandpropagated.Thisisofgreatimportanceforourunderstandingofmanydiseasesofe.g.thekidneys,heart,musclesandnervoussystem.Thatthebody’scellsmustcontainspecificchannelsfortransportingwaterwassuspectedasearlyasthemiddleofthenineteenthcentury.However,itwasnotuntil1988that

PeterAgre

succeededinisolatingamembraneproteinthat,ayearorsolater,herealisedmustbethelong-sought-afterwaterchannel.Thisdecisivediscoveryopenedthedoortoawholeseriesofbiochemical,physiologicalandgeneticstudiesofwaterchannelsinbacteria,plantsandmammals.Today,researcherscanfollowindetailawatermoleculeonitswaythroughthecellmembraneandunderstandwhyonlywater,notothersmallmoleculesorions,canpass.Theothertypeofmembranechannelwhichisthesubjectofthisyear’sPrizeistheionchannel.

RoderickMacKinnon

surprisedthewholeresearchcommunitywhenin1998hewasabletodeterminethespatialstructureofapotassiumchannel.Thankstothiscontributionwecannow“see”ionsflowingthroughchannelsthatcanbeopenedandclosedbydifferentcellularsignals.Theionchannelsareimportantfor,amongotherthings,thefunctionofthenervoussystemandthemuscles.Whatiscalledtheactionpotentialofnervecellsisgeneratedwhenanionchannelonthesurfaceofanervecellisopenedbyachemicalsignalsentfromanadjacentnervecell,whereuponanelectricalpulseispropagatedalongthesurfaceofthenervecellthroughtheopeningandclosingoffurtherionchannelsinthecourseofafewmilliseconds.Thisyear’sPrizeillustrateshowcontemporarybiochemistryreachesdowntotheatomiclevelinitsquesttounderstandthefundamentalprocessesoflife.Readmoreaboutthisyear’sprizeInformationforthePublic

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PeterAgre,born1949(54years)inNorthfield,Minnesota(UScitizen).MedicalDoctor1974atJohnsHopkinsUniversitySchoolofMedicine,Baltimore,USA.ProfessorofBiologicalChemistryandProfessorofMedicineatJohnsHopkinsUniversitySchoolofMedicine,Baltimore,USA.RoderickMacKinnon,born1956(47years).GrewupinBurlingtonoutsideBoston,USA(UScitizen).MedicalDoctor1982atTuftsMedicalSchool,Boston,USA.ProfessorofMolecularNeurobiologyandBiophysicsatTheRockefellerUniversityinNewYork,USA.Prizeamount:SEK10million,willbesharedequallyamongtheLaureates.Contactpersons:MalinLindgren,Informationofficer,Phone+4686739522,+46709886004,

malin@kva.se,EvaKrutmeijer,HeadofInformation,Phone+4686739595,+46709846638,

evak@kva.se2003年诺贝尔化学奖被授予“有关细胞膜通道的发现”，彼得·阿格里（PeterAgre）一半被授予“水通道的发现”，罗德瑞克·麦金农（RoderickMacKinnon）被一半授予“离子通道的结构和力学研究”。分子通道让我们进入细胞化学我们人类的盐水约占70％。今年的诺贝尔化学奖奖励了两位科学家，他们的发现阐明了盐（离子）和水是如何从人体细胞中运出和运出的。这些发现使我们对肾脏如何从原尿中回收水以及我们的神经细胞中的电信号如何产生和传播有了基本的分子理解。这对于我们了解许多疾病（例如传染病）非常重要。肾脏，心脏，肌肉和神经系统。早在十九世纪中叶，人们就怀疑人体的细胞必须包含特定的输水通道。但是，直到1988年，彼得·阿格里（PeterAgre）才成功分离出膜蛋白，一年左右后，他意识到这必定是人们长期以来一直期待的水道。这一决定性发现为细菌，植物和哺乳动物中水通道的一系列生化，生理和遗传研究打开了大门。如今，研究人员可以详细跟踪水分子通过细胞膜的过程，并了解为什么只有水而不是其他小分子或离子可以通过。本年度奖项的另一类膜通道是离子通道。RoderickMacKinnon在1998年能够确定钾通道的空间结构时，令整个研究界感到惊讶。由于有了这一贡献，我们现在可以“看到”离子流过可以被不同细胞信号打开和​​关闭的通道。离子通道对神经系统和肌肉的功能尤其重要。当神经细胞表面的离子通道被相邻神经细胞发出的化学信号打开时，就会产生所谓的神经细胞动作电位，然后电脉冲通过神经细胞表面沿神经细胞表面传播。在几毫秒内打开和关闭其他离子通道。今年的奖项说明了当代生物化学如何深入了解原子的基本过程。进一步了解今年的奖项公众信息进阶资讯（pdf）链接和进一步阅读彼得·阿格里（PeterAgre），1949年出生（54岁），美国明尼苏达州诺斯菲尔德。1974年，美国巴尔的摩约翰霍普金斯大学医学院医学博士。美国巴尔的摩约翰霍普金斯大学医学院生物化学教授和医学教授。RoderickMacKinnon，生于1956年（47岁）。在美国波士顿以外的伯灵顿长大（美国公民）。美国波士顿塔夫茨医学院的1982年医学博士。美国纽约洛克菲勒大学分子神经生物学和生物物理学教授。TheNobelPrizeinPhysiologyorMedicine1991wasawardedjointlytoErwinNeherandBertSakmann"fortheirdiscoveriesconcerningthefunctionofsingleionchannelsincells."SummaryEachlivingcellissurroundedbyamembranewhichseparatestheworldwithinthecellfromitsexterior.Inthismembranetherearechannels,throughwhichthecellcommunicateswithitssurroundings.Thesechannelsconsistofsinglemoleculesorcomplexesofmoleculesandhavetheabilitytoallowpassageofchargedatoms,thatisions.Theregulationofionchannelsinfluencesthelifeofthecellanditsfunctionsundernormalandpathologicalconditions.TheNobelPrizeinPhysiologyorMedicinefor1991isawardedforthediscoveriesofthefunctionofionchannels.ThetwoGermancellphysiologists

ErwinNeher

and

BertSakmann

havetogetherdevelopedatechniquethatallowstheregistrationoftheincrediblysmallelectricalcurrents(amountingtoapicoampere–10-12A)thatpassesthroughasingleionchannel.Thetechniqueisuniqueinthatitrecordshowasinglechannelmoleculealtersitsshapeandinthatwaycontrolstheflowofcurrentwithinatimeframeofafewmillionthsofasecond.NeherandSakmannconclusivelyestablishedwiththeirtechniquethationchannelsdoexistandhowtheyfunction.Theyhavedemonstratedwhathappensduringtheopeningorclosureofanionchannelwithadiametercorrespondingtothatofasinglesodiumorchlorideion.Severalionchannelsareregulatedbyareceptorlocalizedtoonepartofthechannelmoleculewhichuponactivationaltersitsshape.NeherandSakmannhaveshownwhichpartsofthemoleculethatconstitutethe“sensor”andtheinteriorwallofthechannel.Theyalsoshowedhowthechannelregulatesthepassageofpositivelyornegativelychargedions.Thisnewknowledgeandthisnewanalyticaltoolhasduringthepasttenyearsrevolutionizedmodernbiology,facilitatedresearch,andcontributedtotheunderstandingofthecellularmechanismsunderlyingseveraldiseases,includingdiabetesandcysticfibrosis.WhatHappensInsidetheCell?Insidethecellmembranethereisawell-definedenvironment,inwhichmanycomplexbiochemicalprocessestakeplace.Theinteriorofthecelldiffersinimportantrespectsfromitsoutside.Forexamplethecontentsofpositivesodiumandpotassiumionsandnegativelychargedchlorideionsarequitedifferent.Thisleadstoadifferenceinelectricalpotentialoverthecellmembrane,amountingto0.03to0.1volts.Thisisusuallyreferredtoasthemembranepotential.Thecellusesthemembranepotentialinseveralways.Byrapidlyopeningchannelsforsodiumionsthemembranepotentialisalteredradicallywithinathousandthofasecond.Cellsinthenervoussystemcommunicatewitheachotherbymeansofsuchelectricalsignalsofaroundatenthofavoltthatrapidlytravelalongthenerveprocesses.Whentheyreachthepointofcontactbetweentwocells–thesynapse–theyinducethereleaseofatransmittersubstance.Thissubstanceaffectsreceptorsonthetargetcell,oftenbyopeningionchannels.Themembranepotentialisherebyalteredsothatthecellisstimulatedorinhibited.Thenervoussystemconsistsofaseriesofnetworkseachcomprisedofnervecellsconnectedbysynapseswithdifferentfunctions.Newmemorytracesinthebrainareforexamplecreatedbyalteringthenumberofavailableionchannelsinthesynapsesofagivennetwork.Allcellsfunctioninasimilarway.Infact,lifeitselfbeginswithachangeinmembranepotential.Asthespermmergeswiththeeggcellattheinstantoffertilizationionchannelsareactivated.Theresultantchangeinmembranepotentialpreventstheaccessofotherspermcells.Allcells–forinstancenervecells,glandcells,andbloodcells–haveacharacteristicsetofionchannelsthatenablethemtocarryouttheirspecificfunctions.Theionchannelsconsistofsinglemoleculesorcomplexesofmolecules,thatformsthewallofthechannel–orpore–thattraversesthecellmembraneandconnectstheexteriortotheinteriorofthecell(Figure1Band1D).Thediameteroftheporeissosmallthatitcorrespondstothatofasingleion(0.5-0.6millionthsofamillimetre).Animmediatechangeintheshapeofthemoleculeleadstoeitheranopeningoraclosureoftheionchannel.Thiscanoccuruponactivationofthereceptorpartofthemolecule(Figure1D)byaspecificsignalmolecule.Alternativelyaspecificpartofthemoleculethatsenseschangesinmembranepotentialcanopenorclosetheionchannel.Figure1.RegistrationoftheflowofcurrentthroughsingleionchannelsusingtherecordingtechniqueofNeherandSakmann.

A

schematicallyshowshowaglassmicropipetteisbroughtincontactwiththecell,and

B,usingahighermagnification,apartofthecellmembrane,withionchannels,inclosecontactwiththetipofthepipette.Theinteriorofthepipetteisconnectedtoanelectronicamplifier.

C

showsachannelingreatermagnificationwithitsreceptorfacingtheexteriorofthecellanditsionfilter.

D

showsthecurrentpassingthroughtheionchannelasitopens.NeherandSakmannRecordtheElectricCurrentFlowingThroughaSingleIonChannelIthaslongbeenknownthatthereisarapidionexchangeoverthecellmembrane,butNeherandSakmannwerethefirsttoshowthatspecificionchannelsactuallyexist.Toelucidatehowanionchanneloperatesitisnecessarytobeabletorecordhowthechannelopensandcloses.Thisappearedelusivesincetheioniccurrentthroughasingleionchannelisextraordinarilysmall.Inaddition,thesmallionchannelmoleculesareembeddedinthecellmembrane.NeherandSakmannsucceededinsolvingthesedifficulties.Theydevelopedathinglassmicropipette(athousandthsofamillimeterindiameter)asarecordingelectrode.Whenitisbroughtincontactwiththecellmembrane,itwillformatightsealwiththeperipheryofthepipetteorifice(Figure1A,B).Asaconsequencetheexchangeofionsbetweentheinsideofthepipetteandtheoutsidecanonlyoccurthroughtheionchannelinthemembranefragment(Figure1B).Whenasingleionchannelopens,ionswillmovethroughthechannelasanelectriccurrent,sincetheyarecharged.Througharefinementoftheelectronicequipmentandtheexperimentalconditionstheysucceededinmeasuringthis“microscopical”currentbylaboriousmethodologicaldevelopmentsduringtheseventies(Figure1C).HowDoesanIonChannelOperate?Ionchannelsareofdifferenttypes.Someonlypermittheflowofpositivelychargedsodium,potassiumorcalciumions,othersonlynegativelychargedchlorideions.NeherandSakmanndiscoveredhowthisspecificityisaccomplished.Onereasonisthediameteroftheionchannel,whichisadaptedtothediameterofaparticularion.Inoneclassofionchannels,therearealsotworingsofpositivelyornegativelychargedaminoacids.Theyformanionicfilter(seeFigure1D),whichonlypermitsionswithanoppositechargetopassthroughthefilter.InparticularSakmannthroughacreativeinteractionwithdifferentmolecularbiologistselucidatedhowthedifferentpartsoftheionchannelmolecule(s)operate.NeherandSakmann’sscientificachievementshaveradicallychangedourviewsonthefunctionofthecellandthecontentsoftextbooksofcellbiology.Theirmethodsarenowusedbythousandsofscientistsallovertheworld.TheStudyofSecretoryProcessesNervecells,aswellashormone-producingcellsandcellsengagedinthehostdefence(likemastcells)secretedifferentagents.Theyarestoredinvesiclesenclosedbyamembrane.Whenthecellisstimulatedthevesiclesmovetothecellsurface.Thecellandvesiclemembranesfuseandtheagentisliberated.Themastcellsecreteshistamineandotheragentsthatgiverisetolocalinflammatoryreactions.Thecellsoftheadrenalmedullaliberatethestresshormoneadrenaline,andthebetacellsinthepancreasinsulin.Neherelucidatedthesecretoryprocessesinthesecelltypesthroughthedevelopmentofanewtechniquewhichrecordsthefusionofthevesicle(s)withthecellmembrane.Neherrealizedthattheelectricpropertiesofacellwouldchangeifitssurfaceareaincreasedmakingitpossibletorecordtheactualsecretoryprocess.Throughfurtherdevelopmentsoftheirsophisticatedequipmenttheresolutionfinallypermittedrecordingofeachlittlevesiclefusingwiththecellmembrane.RegulationofIonChannelFunctionNeherandSakmannalsousedtheelectrodepipettetoinjectdifferentagentsintothecell,andtheycouldtherebyinvestigatethedifferentstepsinthesecretoryprocesswithinthecellitself(seeabove).InthiswayanumberofcellularsecretorymechanismshavebeenclarifiedsuchastheroleofcyclicAMP(see

NobelPrizetoSutherland1971)orcalciumions.Forinstance,wenowhaveabetterunderstandingofhowthehormonelevelsinthebloodaremaintainedatacertainlevel.Alsothebasalmechanismsunderlyingthesecretionofinsulinhavebeenidentified.Thelevelofbloodglucosecontrolsthelevelofglucosewithintheinsulin-formingcell,whichinturnregulatestheleveloftheenergyrichsubstanceATP.ATPactsdirectlyonaparticulartypeofionchannelwhichcontrolstheelectricmembranepotentialofthecell.Thechangeofmembranepotentialthenindirectlyinfluencesotherionchannels,whichpermitcalciumionstopassintothecell.Thecalciumionssubsequentlytriggertheinsulinsecretion.Indiabetestheinsulinsecretionisoutoforder.CertaindrugscommonlyusedtostimulateinsulinsecretionindiabetesactdirectlyontheATP-controlledionchannels.Manyotherdiseasesdependentirely,orpartially,onadefectregulationofionchannels,andanumberofdrugsactdirectlyonionchannels.Manypathologicalmechanismshavebeenclarifiedduringtheeightiesthroughionchannelstudies,forinstancecysticfibrosis(clorideionchannels),epilepsy(sodiumandpotassiumionchannels),severalcardio-vasculardiseases(calciumionchannels),andneuro-musculardisorderslikeLambert-Eatonsdisease(calciumionchannels).WiththehelpofthetechniqueofNeherandSakmannitisnowpossibletotailormakedrugs,toachieveanoptimaleffectonparticularionchannelsofimportanceinagivendisease.Drugsagainstanxietyactforinstanceoncertaininhibitoryionicchannelsinthebrain.Alcohol,nicotineandotherpoisonsactonyetothersetsofionchannels.Insummary,NeherandSakmann’scontributionshavemeantarevolutionforthefieldofcellbiology,fortheunderstandingofdifferentdiseasemechanisms,andopenedawaytodevelopnewandmorespecificdrugs.ReferencesAlbertsetal.:TheMolecularBiologyoftheCell.GarlandPress,1990,2ndedition,pp.156,312-326,1065-1084.Grillner,S.I:N.Calder(ed.).ScientificEurope.FoundationScientificEurope,1990.Grillner,S.&Hökfelt,T.:Svindlandesnabbutvecklingpräglarneurovetenskapen.Läkartidningen1990,

87,2777-2786.Rorsman,P.&Fredholm,B.B.:Jonkanaler–molekylärbakgrundtillnervtransmission.Läkartidningen1991,

88,2868-2877.1991年的诺贝尔生理学或医学奖是由ErwinNeher和BertSakmann共同授予的，“因为他们发现了细胞中单个离子通道的功能”。摘要每个活细胞被膜包围，该膜将细胞内的世界与其外部隔开。在该膜中存在通道，细胞通过通道与周围环境进行通讯。这些通道由单个分子或分子的复合物组成，并具有使带电原子（即离子）通过的能力。在正常和病理条件下，离子通道的调节会影响细胞的寿命及其功能。1991年诺贝尔生理学或医学奖因发现离子通道的功能而获得。两位德国细胞生理学家ErwinNeher和BertSakmann共同开发了一种技术，该技术可以记录流经单个离子通道的极小的电流（相当于10安培至12A的电流）。该技术的独特之处在于它可以记录单个通道分子如何改变其形状，并以此方式在几百万分之一秒的时间范围内控制电流的流动。Neher和Sakmann通过他们的技术最终确定了离子通道的存在及其功能。他们证明了在打开或关闭直径对应于单个钠或氯离子直径的离子通道期间会发生什么。几个离子通道由位于通道分子一部分上的受体调节，该受体在激活后会改变其形状。Neher和Sakmann已表明构成“传感器”的分子的哪些部分以及通道的内壁。他们还展示了通道如何调节带正电或带负电的离子的通过。在过去的十年中，这一新知识和新分析工具革新了现代生物学，促进了研究，并有助于理解包括糖尿病和囊性纤维化在内的几种疾病的细胞机制。细胞内部会发生什么？在细胞膜内部有一个明确的环境，其中发生了许多复杂的生化过程。电池的内部在重要方面与外部不同。例如，正钠离子和钾离子以及带负电荷的氯离子的含量是完全不同的。这导致整个细胞膜上的电位差达到0.03至0.1伏。通常将其称为膜电位。细胞以多种方式利用膜电位。通过迅速打开钠离子通道，膜电势在千分之一秒内发生根本变化。神经系统中的细胞通过十分之一伏特的电信号相互通讯，这些信号迅速沿着神经过程传播。当它们到达两个细胞（突触）之间的接触点时，它们会诱导释放递质。该物质通常通过打开离子通道来影响靶细胞上的受体。从而改变膜电位，从而刺激或抑制细胞。神经系统由一系列网络组成，每个网络都由具有不同功能的突触连接的神经细胞组成。例如，通过改变给定网络突触中可用离子通道的数量，可以创建大脑中的新记忆轨迹。所有单元的功能均相似。实际上，生命本身始于膜电位的变化。受精时，随着精子与卵细胞融合，离子通道被激活。膜电位的最终变化阻止了其他精子细胞的进入。所有细胞（例如神经细胞，腺细胞和血细胞）都有一组独特的离子通道，使它们能够执行其特定功能。离子通道由单个分子或分子的复合物组成，形成通道的壁或孔，该壁穿过细胞膜并连接细胞的外部与内部（图1B和1D）。孔的直径是如此之小，