Name:_____KEY_________________________

Page1

Neuroscience201AExam“Key”,October7,2014

Question#1 7.5pts

Considerasphericalneuronwithadiameterof20µmandarestingpotentialof-70mV.Ifthenetnegativityontheinsideofthecell(allofthe“excess”anions)wereuniformlydistributedwithinthevolumeofthecellratherthanbeing“glommed”ontotheinnersurfaceoftheplasmamembrane,whatwouldtheconcentrationoftheseionsbe(inM)?Assumethattheseareallunivalentions.Assumethatthecapacitanceoftheplasmamembraneisthestandard1µF/cm2.

Name:_____KEY_________________________

Page2

Question#2 12.5pts

Imaginethatyouhaveasquidneuronwithasinglepopulationofleakchannels,whicharecationchannelswithpermeabilitytoonlyNa+andK+.Theconcentrationsofpermeantionsoneachsideofthemembraneareasfollows:

Ion Na+ K+

Outside 550mM 20mM

Inside 55mM 440mM

a) Iftherestingmembranepotentialofthecellis-63mV,calculategNa/gKfortheleakchannels.(Youareencouragedtouseconductancesforthisproblem(notpermeabilities).

b) Ifthesquidneuronissphericalwithadiameterof200µm,iftheleakconductanceofitsmembraneis0.3mS/cm2,andiftheconductanceofasingleleakchannelis10pS,calculatetheaveragedensityofleakchannelsinthemembrane,as#perµm2.

Name:_____KEY_________________________

Page3

Question#3 12.5pts

Theabovefigureillustratesasetofcurrentresponses(a,lowertraces)tovoltagesteps(a,toptraces)forapopulationofchannelsinaneuronwithphysiologicalsolutionsbathingtheinsideandoutsideofthecell.(Conventionalgraphics–inwardcurrentisdownwardandnegativeinsign).Thereareleakchannelsinthiscell’smembraneinadditiontothevoltagedependentchannels.

a) Whatistheleakageconductanceofthiscell(theconductanceattributabletoalloftheleakchannels)?Assumethattherestingpotentialis-50mV.Theleakchannelsaretheonesopenatrest.Ifyoustepthemembranepotentialtoavaluedifferentthanrest(rest=Erevfortheleakchannels),thenyouwillseeanimmediatecurrent(immediatebecausethechannelsarealreadyopen).Thearrowin(a)pointstothiscurrent.Thecurrentisabout-0.075x10-9A,andthedrivingforce,Vm–Erev,is-140-(-50)mV=-90mV.FromOhm’slaw,gL=isabout0.8nS.ThisisaLOTsmallerthantheconductanceduetothecationchannelsthatopeninresponsetohyperpolarization(whichcanbecalculatedfromtheslopeofthestraightlinein(b)),whichisabout13nS.

b) InpartbisshownanIVcurvefortheearlycurrentafterthepotentialissteppedfrom-140mVtotheindicatedvalueinthegraph.Whatisthereversalpotentialforthischannel?Towhationorionsdoyoususpectthatthischannelispermeable?Howwouldyoutestyourhypothesis?Ifyouthinkthatthischannelispermeabletomultipleions,howwouldyoumeasurethepermeabilityratios(P1:P2,where1and2representdifferentions).Reversalpotentialisabout-35mV.Thisdoesnotmatchtheequilibriumpotentialforanysingleion,exceptperhapsthatforchloridewhenthechloridetransportersarenotyetfullyexpressedduringearlydevelopment.Thebestguessisthatit’sacationchannelwithaslightlydifferentgNa/gKthantheAMPA

Name:_____KEY_________________________

Page4

ornicotinicreceptorchannelsthatwehavediscussed.Howdoyoutestwhetheranyparticularhypothesisaboutthepermeabilityofanionchanneliscorrect?YouchangetheconcentrationofthehypothesizedpermeantionsononeortheothersideofthemembraneandlooktoseeifthisshiftsErevforthechannel.Ifachannelispermeabletoasingleion,thena10-foldchangeintheconcentrationofthationoneithersideofthemembranewillproducea~60mVshiftinErev.If,alternatively,thechannelispermeabletomultipleions,suchassodiumandpotassium,thena10-foldchangeinoneofthemwillproducelessthana~60mVshiftinErev.(Infact,thesumofthechangestoa10-foldchangeineachion’sconcentrationgradientwillbe~60mV.)NotethatbyclampingthecellatENaandlookingforoutwardcurrentsdoesNOTguaranteethatthesecurrentsarepotassiumcurrents,andcorrespondingly,clampingatEKdoesnotinsurethattheremaininginwardcurrentsaresodiumcurrents.You’vegottochangeconcentrationsandlookforshiftsinErevtomakethisassessment.Onceyouhaveestablishedwhichionsflowacrossthechannel,youcanusetheGHKequation(PorGversion)tocalculatetheratioofpermeabilitiesorconductances.

Name:_____KEY_________________________

Page5

Question#4 15pts

Thefollowingfigureistakenfromapaperonvoltage-dependentcalciumchannelsandshowsthepeakcurrent-voltagerelationforasinglepopulationofchannels.

a) Datashownassquareswerecollectedundercontrolconditions.Fromthesedata,constructacurveofpeakconductancevs.voltageforthischannel,overtherangeof-60mVto+60mV.

b) Thedatashownincircleswerecollectedusingadrug(the“circledrug”).Provideaplausiblehypothesisaboutwhatthe“circledrug”doestoaltertheIVcurveforthischannel.Whatsortofevidence(obtainedinanexperimentofyourchoosing)wouldsupportthishypothesis?

Thedatashownintriangleswerecollectedinthepresenceofadifferentdrug(the“triangledrug”).

c) Thefiguresupportsthepossibilitythatoneofthetwodrugs(circle,triangle)alterstheionselectivityofthechannels.Whichdrugisthis,andonwhatdoyoubaseyourassertion?

a) HeretheideaistodowhatH&Hdidintheirsecond1952paper(116:449-472)andconvertfromcurrenttoconductanceusingOhm’sLaw.Fromthedata,wecanassumethatErevforthechannelis+50mV.Thedataisbelow.NotethatthecloseryougettoErev,thegreatertheerror,sinceyouaredividingbyanumber,thedrivingforce,thatisapproachingzero.Thepointat+60mVhasalowerconductancethatthoseat+30-+40mV.Thisisnotlikelytosignifythatfewerchannelsopenwithastrongerdepolarization(whichwouldrequireamuchmorecomplicatedmodelforhowthechannelworks)butratherjustthatthere’smoreerror.Mostconductancecurveslikethisonewillreachamaximumandstaytherewithstrongerandstrongerdepolarizations(orhyperpolarizations,ifthat’swhatopensthechannel).

Name:_____KEY_________________________

Page6

b) ThecircledrugdoesnotchangeErev,butitgreatlyincreasestotalcurrent.Eithertherearemorechannelsopening(morelikely),ortheconductanceoftheopenchannelislarger(lesslikely,sincethismightbeexpectedtochangeselectivityandallowmorepotassiumtopass,whichwouldshiftErevtomorenegativevalues).Onewaytogetmorechannelstoopeninresponsetoadepolarizationistoremoveinactivation.Ifthisdrugremovedinactivationofsomeofthecalciumchannels,morewouldbeavailabletoopenandmorewouldopen,withoutnecessarilychangingtheactivationsensitivityofthechannels.Onewaytotestforthispossibilityistoseeifyoucaneliminatetheeffectofthedrugbydeliveringapre-pulsetoalargenegativemembranepotential,whichshouldremoveallinactivationfromthechannels.Uponsteppingthemembranepotentialto,say,0mV,youwillgetaverylargecurrent,andthecircledrugshouldnotfurtherenhancethecurrent.Anotherpossibilityisthatthedrugmakesthechannelmoresensitivetovoltage,suchthatmoreopeninresponsetoadepolarizationofanymagnitude.Thiswouldhelpexplaintheapparentshiftinvoltagesensitivityofthechannel,suchthattheclamppotentialatwhichthecurrentpeaksisshiftedfrom+10mVto0mV.However(andImightbewrongaboutthis),simplyincreasingthevoltagesensitivitywouldnotbeexpectedtoincreasethemaximalconductance,andinthisinstance,themaximalconductanceis2-3xtimesgreaterinthepresenceofthecircledrug.Theotherpossibilityisthatthecircledrugincreasestheconductanceofasinglechannel;thesamenumberofchannelsopen,buteachpassesmorecurrent.Onechallengeistodothiswithoutchangingselectivity,whichisnotchanged(Erev).If,somehow,Naturecouldmanagethis,thenyouwouldexpecttofindthattheopenchannelconductancewouldbeincreased.c)Thetriangledrugislikelytobetheonethatalterstheselectivityofthechannel,sinceErevischangedfromabout+50mVtoabitlessthan+40mV.Thisdrugmight,forexample,reducethecalciumselectivityofthechannelandintroducesomepotassium(out)movement.Iftheconductanceofanopenchannelwereconstant,it’sunlikelythatthemodestchangeinselectivitycouldexplainthereductionofcurrentthatisobserved.

Name:_____KEY_________________________

Page7

Question#5 12.5pts

Thefollowingpassagerecentlyappearedinthe“News&Views”sectionofoneofthejournalswithaonewordname.“Gliomas,acommonbraintumor,frequentlyinduceepilepticseizuresastheygrow,butitisnotclearwhy.LastNameetal.(XXXX)showthattheepilepticactivityinthebrainaroundgliomashappensbecauseneuronsrespondanomalouslytotheneurotransmitterg-aminobutyricacid(GABA).UsuallyGABAturnsneuronsoff,butwhengliomasarenearby,itturnsthemoninstead.”

WhatdoyouhypothesizethatgliomasaredoingtoneuronsthatwouldallowGABAtodepolarizethemenoughtofirethem?Describeanexperimentaltestofyourprediction.Assumethatyoucanwholecellrecord/clampfromneuronsinsliceswithgliomasnearbyandthatyoucanactivateGABAergicinputsontothesecells.

NearlyallofyousuggestedthatGABAisactingbydrivingthemembraneofneuronsneargliomastowardsaErevthatismorepositivethaninthecontrolcase:morepositivethanthreshold.Therearetwopossibilitieshere:thefirstisthatGABAgatesaCl-withaEClthatislessnegativethannormal.ThisistheactualexplanationfortheworkcitedintheNews&Viewsarticle.IfEClislessnegativethannormal,thentheCl-gradientmustbemoreshallowthannormal,andthismayoccureitherbecauseintracellularCl-ishigherthannormalorbecauseextracellularislowerthannormal.Thesecondpossibilityisnotverylikely,sinceitwouldrequirethatinthesamegeneralvolumetherearetwodifferentconcentrationsofextracellularCl-.MorelikelyisthepossibilitythatintracellularCl-iselevated,andthismayoccurbecausethechloridepotassium(KCC2)symporter(which“carries”Cl-outofthecellusingtheK+electrochemicalgradient)isinhibitedordownregulated.Howwouldyoutestforthis?ThesimplestwaywouldbetoactivatetheGABAsynapticinputwhileclampingthepostsynapticcellandtomeasureErevfortheinput.EGABA(Erev)shouldbemorepositiveforneuronsnearergliomasintheslice.ButhowwouldyouknowthatthiswasbecauseofachangeinEClandnotsimplyachangeintheselectivityoftheGABAreceptor(thesecondpossibleexplanationforwhyErevmightbemorepositiveneargliomas)?ThecleanestapproachherewouldbetomeasureErevbeforeandafterchangingtheCl-equilibrium(Nernst)potential.IfthechannelisselectiveentirelyforCl-,thena10-foldchangeinEClwillproducea~60mVshilftinErev(GABA).YoucouldalsoproposemeasuringCl-concentrationdirectlyinsidethecell,andIthinkthattherearesomefluorescentindicatorsdyesthatallowyoudotothat(notsurewhethertheyareproventobeusefulinthissituation).IfErevismoredepolarizedbecausethechannelisnolongerselectivetoCl-buthascationorsodiumpermeabilityaswell,thenwhenyouchangeECl-by10fold,youshouldgetlessthana60mVshiftinErev.Likewise,inthiscaseyouwillgetashiftinErevwhenyouchange,forexample,ENa.

NotethatitwouldbedifficulttoincreaseextracellularCl-aboveitsnormallevel,sincetheoutsideofthecellisapproximatelyisotonicNaCl:anadditionalincreasewouldproduceahypertonicsolutionandmakethecellswell.

Name:_____KEY_________________________

Page8

Question#6 10pts

Thefigureabove,takenfromtheslidesetonsynaptictransmission,illustratesanexampleofaresultinwhichN,thephysiologicallymeasurednumberofreleasablequanta,wasequaltothenumberofsynapticboutons(eachofwhichisthoughttocontainasingleactivezone).

Thedataaboveisinterpretedassupportingaunivesicularreleasemodelforthissynapse.

a) Analternativepossibleexplanationforthedataaboveisthatreleaseismultivesicular,butthatasinglequantumoftransmittersaturatestheAMPAreceptorsatthesynapticsite.Thedatashownontheleft(Fig.4)argueagainstthispossibility.Explainwhy.

b) Whatsourcesofintersitequantalvariancearelikelytobeatplayinthisexperimentalarrangement?

Name:_____KEY_________________________

Page9

Suggestedanswertoquestion#6(previouspage):

(a) Thedatashowninthetoppartofthepreviouspageisequallycompatiblewithaunivesicularmodelofrelease,withorwithoutsaturationofAMPARswithasinglequantumoftransmitter,orwithamultivesicularmodelofrelease,withsaturationofAMPARswithasinglequantumoftransmitter.Whatresultinthelowerfiguresuggeststhatthereisnotmultivesicularrelease?Thefindingthattheefficacy(%block)withDGGisthesameattwodifferentextracellularcalciumconcentrations,whichproduceverydifferentsizedEPSCs,suggeststhatreleaseisnotmultivesicular.Haditbeenmultivesicular,onewouldhaveexpectedDGGtomoreeffectivelybetteratthelowerextracellularcalciumconcentration.

(b) Wecoveredseveralpossiblesourcesofintersitequantalvariabilityinclass,includingvariabilityintheamountofglutamatepackagedinavesicle(assumingnosaturationofgluRswithaquantum),variabilityinthenumberofgluRspostsynapticallyorintheirproperties,variabilityintheanatomyofthesynapse(escaperoutesforglu,glialcellsnearbywithglutransporters).Togetfullcredit,youhadtoaddthatanothersourceofvariabilityisthedistancebetweentherecordingsite(thecellbody)andeachofthesites(seefigure,topofpreviouspage,lowerleftpanel).Sitesthataremoredistantmayproducesmallerandslowerresponses,ormaybenot(nowthatyouknowaboutthingslikesynapticscaling).

NotethatvariationsinNorparenotsourcesofquantalvariability,sincewearetalkingaboutsinglequantalresponsesandnotabouttrial-to-trialvariabilityinthenumberofquantareleased.

Question#7

(Pleasewriteyourresponsetothisquestiononaseparatepage,ifyouarecompletingtheexaminpaperformat.)

Howarehighselectivityandhighrateoftransportthroughionchannelsachievedsimultaneously?DoyouthinkNa+canpermeatethroughaCa2+-selectiveionchannel?Ifyes,underwhatconditions?Pleaseexplainyouranswers.

15pts

Highselectivityofionchannelsisachievedwithhigh-affinitybindingsitesforthepermeantionintheselectivityfilter.Thehighrateoftransportisachievedbyplacingseveralsuchbindingsitesincloseproximitytoeachother,sothatwhenallormajorityofthebindingsitesareoccupiedbypermeantions,theelectrostaticrepulsionbetweentheionsreducesbindingaffinityandsignificantlyshortensthetimetheionsdwellinpore.

Na+canpermeatethroughCa2+selectivechannelsintheabsenceofCa2+.UndertheseconditionsNa+canoccupyCa2+-bindingsitesintheporeandgothroughthechannel.WhenCa2+ispresent,itoutcompetesNa+anddoesnotallowittobindinthepore.

Name:_____KEY_________________________

Page10

Question#8

(Pleasewriteyourresponsetothisquestiononaseparatepage,ifyouarecompletingtheexaminpaperformat.)

WhatistheroleofS1,S2andS3transmembranehelicesofthevoltage-gatedionchannelsinthemechanismofvoltagesensitivity?Doyouthinkitwouldbepossibletoconstructasimplervoltage-sensordomainthatwouldconsistofS4domainonly?Pleaseexplainyouranswers.

15pts

S4helixhasseveralpositivelychargedaminoacidresiduesthatmustbestabilizedwithinthehydrophobicenvironmentofthelipidmembrane.S1–S3helixesprovidenegativelycharged/polarresiduesthatformatransmembranetranslocationpathwaythatallowsS4helixtoexistandmovewithinthelipidmembrane.ThereareadditionalbenefitsofS1-S3helixes.SpecificstatesoftheS4helix(suchasopenandclosed)canbestabilizedascomparedto“intermediate”states.Also,S1-S3helixesformafoundationthatS4helixusestoapplyforcetotheporedomain.AllthefactorsmentionedabovemakeitveryunlikelythataVSDcanbeconstructedofS4helixonly.