(7.1)-拓展知識(shí)（1）離子通道與膜片鉗技術(shù)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年諾貝爾化學(xué)獎(jiǎng)被授予“有關(guān)細(xì)胞膜通道的發(fā)現(xiàn)”，彼得·阿格里（PeterAgre）一半被授予“水通道的發(fā)現(xiàn)”，羅德瑞克·麥金農(nóng)（RoderickMacKinnon）被一半授予“離子通道的結(jié)構(gòu)和力學(xué)研究”。分子通道讓我們進(jìn)入細(xì)胞化學(xué)我們?nèi)祟惖柠}水約占70％。今年的諾貝爾化學(xué)獎(jiǎng)獎(jiǎng)勵(lì)了兩位科學(xué)家，他們的發(fā)現(xiàn)闡明了鹽（離子）和水是如何從人體細(xì)胞中運(yùn)出和運(yùn)出的。這些發(fā)現(xiàn)使我們對(duì)腎臟如何從原尿中回收水以及我們的神經(jīng)細(xì)胞中的電信號(hào)如何產(chǎn)生和傳播有了基本的分子理解。這對(duì)于我們了解許多疾病（例如傳染?。┓浅Ｖ匾?。腎臟，心臟，肌肉和神經(jīng)系統(tǒng)。早在十九世紀(jì)中葉，人們就懷疑人體的細(xì)胞必須包含特定的輸水通道。但是，直到1988年，彼得·阿格里（PeterAgre）才成功分離出膜蛋白，一年左右后，他意識(shí)到這必定是人們長(zhǎng)期以來一直期待的水道。這一決定性發(fā)現(xiàn)為細(xì)菌，植物和哺乳動(dòng)物中水通道的一系列生化，生理和遺傳研究打開了大門。如今，研究人員可以詳細(xì)跟蹤水分子通過細(xì)胞膜的過程，并了解為什么只有水而不是其他小分子或離子可以通過。本年度獎(jiǎng)項(xiàng)的另一類膜通道是離子通道。RoderickMacKinnon在1998年能夠確定鉀通道的空間結(jié)構(gòu)時(shí)，令整個(gè)研究界感到驚訝。由于有了這一貢獻(xiàn)，我們現(xiàn)在可以“看到”離子流過可以被不同細(xì)胞信號(hào)打開和??關(guān)閉的通道。離子通道對(duì)神經(jīng)系統(tǒng)和肌肉的功能尤其重要。當(dāng)神經(jīng)細(xì)胞表面的離子通道被相鄰神經(jīng)細(xì)胞發(fā)出的化學(xué)信號(hào)打開時(shí)，就會(huì)產(chǎn)生所謂的神經(jīng)細(xì)胞動(dòng)作電位，然后電脈沖通過神經(jīng)細(xì)胞表面沿神經(jīng)細(xì)胞表面?zhèn)鞑ァＴ趲缀撩雰?nèi)打開和關(guān)閉其他離子通道。今年的獎(jiǎng)項(xiàng)說明了當(dāng)代生物化學(xué)如何深入了解原子的基本過程。進(jìn)一步了解今年的獎(jiǎng)項(xiàng)公眾信息進(jìn)階資訊（pdf）鏈接和進(jìn)一步閱讀彼得·阿格里（PeterAgre），1949年出生（54歲），美國(guó)明尼蘇達(dá)州諾斯菲爾德。1974年，美國(guó)巴爾的摩約翰霍普金斯大學(xué)醫(yī)學(xué)院醫(yī)學(xué)博士。美國(guó)巴爾的摩約翰霍普金斯大學(xué)醫(yī)學(xué)院生物化學(xué)教授和醫(yī)學(xué)教授。RoderickMacKinnon，生于1956年（47歲）。在美國(guó)波士頓以外的伯靈頓長(zhǎng)大（美國(guó)公民）。美國(guó)波士頓塔夫茨醫(yī)學(xué)院的1982年醫(yī)學(xué)博士。美國(guó)紐約洛克菲勒大學(xué)分子神經(jīng)生物學(xué)和生物物理學(xué)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.&H?kfelt,T.:Svindlandesnabbutvecklingpr?glarneurovetenskapen.L?kartidningen1990,

87,2777-2786.Rorsman,P.&Fredholm,B.B.:Jonkanaler–molekyl?rbakgrundtillnervtransmission.L?kartidningen1991,

88,2868-2877.1991年的諾貝爾生理學(xué)或醫(yī)學(xué)獎(jiǎng)是由ErwinNeher和BertSakmann共同授予的，“因?yàn)樗麄儼l(fā)現(xiàn)了細(xì)胞中單個(gè)離子通道的功能”。摘要每個(gè)活細(xì)胞被膜包圍，該膜將細(xì)胞內(nèi)的世界與其外部隔開。在該膜中存在通道，細(xì)胞通過通道與周圍環(huán)境進(jìn)行通訊。這些通道由單個(gè)分子或分子的復(fù)合物組成，并具有使帶電原子（即離子）通過的能力。在正常和病理?xiàng)l件下，離子通道的調(diào)節(jié)會(huì)影響細(xì)胞的壽命及其功能。1991年諾貝爾生理學(xué)或醫(yī)學(xué)獎(jiǎng)因發(fā)現(xiàn)離子通道的功能而獲得。兩位德國(guó)細(xì)胞生理學(xué)家ErwinNeher和BertSakmann共同開發(fā)了一種技術(shù)，該技術(shù)可以記錄流經(jīng)單個(gè)離子通道的極小的電流（相當(dāng)于10安培至12A的電流）。該技術(shù)的獨(dú)特之處在于它可以記錄單個(gè)通道分子如何改變其形狀，并以此方式在幾百萬分之一秒的時(shí)間范圍內(nèi)控制電流的流動(dòng)。Neher和Sakmann通過他們的技術(shù)最終確定了離子通道的存在及其功能。他們證明了在打開或關(guān)閉直徑對(duì)應(yīng)于單個(gè)鈉或氯離子直徑的離子通道期間會(huì)發(fā)生什么。幾個(gè)離子通道由位于通道分子一部分上的受體調(diào)節(jié)，該受體在激活后會(huì)改變其形狀。Neher和Sakmann已表明構(gòu)成“傳感器”的分子的哪些部分以及通道的內(nèi)壁。他們還展示了通道如何調(diào)節(jié)帶正電或帶負(fù)電的離子的通過。在過去的十年中，這一新知識(shí)和新分析工具革新了現(xiàn)代生物學(xué)，促進(jìn)了研究，并有助于理解包括糖尿病和囊性纖維化在內(nèi)的幾種疾病的細(xì)胞機(jī)制。細(xì)胞內(nèi)部會(huì)發(fā)生什么？在細(xì)胞膜內(nèi)部有一個(gè)明確的環(huán)境，其中發(fā)生了許多復(fù)雜的生化過程。電池的內(nèi)部在重要方面與外部不同。例如，正鈉離子和鉀離子以及帶負(fù)電荷的氯離子的含量是完全不同的。這導(dǎo)致整個(gè)細(xì)胞膜上的電位差達(dá)到0.03至0.1伏。通常將其稱為膜電位。細(xì)胞以多種方式利用膜電位。通過迅速打開鈉離子通道，膜電勢(shì)在千分之一秒內(nèi)發(fā)生根本變化。神經(jīng)系統(tǒng)中的細(xì)胞通過十分之一伏特的電信號(hào)相互通訊，這些信號(hào)迅速沿著神經(jīng)過程傳播。當(dāng)它們到達(dá)兩個(gè)細(xì)胞（突觸）之間的接觸點(diǎn)時(shí)，它們會(huì)誘導(dǎo)釋放遞質(zhì)。該物質(zhì)通常通過打開離子通道來影響靶細(xì)胞上的受體。從而改變膜電位，從而刺激或抑制細(xì)胞。神經(jīng)系統(tǒng)由一系列網(wǎng)絡(luò)組成，每個(gè)網(wǎng)絡(luò)都由具有不同功能的突觸連接的神經(jīng)細(xì)胞組成。例如，通過改變給定網(wǎng)絡(luò)突觸中可用離子通道的數(shù)量，可以創(chuàng)建大腦中的新記憶軌跡。所有單元的功能均相似。實(shí)際上，生命本身始于膜電位的變化。受精時(shí)，隨著精子與卵細(xì)胞融合，離子通道被激活。膜電位的最終變化阻止了其他精子細(xì)胞的進(jìn)入。所有細(xì)胞（例如神經(jīng)細(xì)胞，腺細(xì)胞和血細(xì)胞）都有一組獨(dú)特的離子通道，使它們能夠執(zhí)行其特定功能。離子通道由單個(gè)分子或分子的復(fù)合物組成，形成通道的壁或孔，該壁穿過細(xì)胞膜并連接細(xì)胞的外部與內(nèi)部（圖1B和1D）?？椎闹睆绞侨绱酥?，