SingleChannelAnalysisandChannelopathies

JimHuettner

LectureOverview

•  Recordingsinglechannels•  Transitionsbetweenstates•  Exponentialdecay•  Analyzingchannelrecordings•  Hodgkin-Huxleykinetics•  Ballandchaininactivation•  Ionchannelandtransporterpathologies

formingatightseal

patch-clampconfigurations

channelsexistinatleast2states

α

β

theoryofanalysis

Radioactive Decay – Macroscopic Kinetics

X → Y

α exponential function

t / τ

0 1 2 3 4 5

X (t)

0.0

0.2

0.4

0.6

0.8

1.0 X(t) = X0e-t / τ

= X0e-α t

X(τ) = 0.368 * X0

dX / dt = - α X

X(t) = X0 e - α t

α = 1 / τ

meanlifetime=timeconstantfordecay

exponential function

t / τ

0 1 2 3 4 5

X (t)

0.0

0.2

0.4

0.6

0.8

1.0

lifetime histogram

time (lifetime duration)

# of

eve

nts

at a

par

ticul

ar li

fetim

eX0 = 1

X(t) = X0e-t/τ = X0e-αt

X(t) = 0.368 X0 when t = τ

analyzingsingleionchannels

open time histogram

open time

# of

eve

nts

at a

par

ticul

ar li

fetim

e

τ open = 1 / β = mean open time

τ open

closed time histogram

closed time

τ closed = 1 / α = mean closed time

τ closed

time

curr

ent

exponentialdistributionoflifetimes

mean open time = τopen = 1 / α = 1 / closing rate constant β

α C = O β

k1 A = B k-1

Po = O/(O+C) = α/(α+β) [3]

Ot = -(O∞ - Oo)e-t / τ + O∞ [4]

τ = 1/(α+β) [5]

twostatemodelanalysis

exponentialrelaxation

Startingwithallchannelsclosed,thefractionofopenchannelsincreasesexponentiallywithTau=1/(α + β) andmaximalopenfraction=α / (α + β)

threestatemodelanalysis

α1 C O C1 2 β ια2

ιγ

τo themeanopentime=1/(β + γ)

complications

•  LowPopen–difficulttoknowifyouareanalyzingasinglechannel

•  Lowconductance–fluctuationanalysisallowsestimationofunitaryconductanceandmeanopentime

•  Subconductancelevels

Premkumaretal.(1997)JGenPhysiol109:181-189

NMDA receptor with pore loop mutation

Hodgkin Huxley kinetics •  HH modeled Na current

as a product of exponentials

•  Three independent activation gates (m) and one independent inactivation gate (h)

•  Compare this to the three state model where transit to C2 only occurs once the channel has opened

ballandchainmodelofinactivation

See: Armstrong CM, Bezanilla F. (1977) J Gen Physiol. 70:567-90.

Long et al., (2005) Science 305:897-903

Long et al., (2005) Science 305:897-903

IonChannelandTransporterPathologiesDiseasesorsyndromescausedbyabnormaloperation,distributionorregulationofchannelsortransporters•  gainorlossoffunctionmutationsinionchannelgenes(Channelopathies)

•  gainorlossoffunctionmutationsinauxiliarysubunits

•  autoantibodiesagainstchannelprotein•  changesinassembly,traffickingormodulation

OnlineMendelianInheritanceinMan(https://omim.org/)

inheritedordenovoInheritedchannelopathiesarerelativelyrare•  exception:cysticfibrosis–alungdiseaseinvolvingchloridechannelmutations

•  1/2000areaffected1/20arecarriers

•  ΔF508–deletionof3basepairsencodingPhe508underlies2/3ofcasesworldwide>90%ofcasesintheU.S.

Zhangetal.(2017)Cell170:483-491

geneticheterogeneitymutationstodifferentgenessimilarphenotypes

example:mutationstoatleast11differentgenesaffecttheQTinterval•  LongQTsyndrome:KCNQ1,KCNH2,

KCNE1,KCNE2,KCNJ2,KCNJ5(LoF)CACNA1C,SCN5A,SCN4B(GoF)

•  ShortQTsyndrome:CACNA1C,CACNB2,CACNA2D1(LoF)KCNH2,KCNQ1,KCNJ2(GoF)

Kim(2014)KoreanJPediatrics.57:1-18.

dominantvsrecessivedifferentmutationstothesamegenemaybedominantorrecessive•  LoFrecessive;GoFlikelytobedominant•  Haploinsufficiency–lossofonealleleshouldreduceproteinlevelby~50%,whichmaybeenoughtoalterphenotype

•  Dominant-negativemutations–themutantcopyadverselyimpactsthewildtypecopy(multimericproteins)

KATPchannelsandinsulinsecretionElevatedbloodglucoseleadstoareductioninKATPchannelactivityinpancreaticβcells,increasingexcitabilityandpromotinginsulinsecretion

•  LoFmutantsexhibithyperinsulinemiaandchroniclowbloodglucose

•  GoFmutantsareneonataldiabeticswithlowinsulinreleaseandelevatedbloodglucose

ColinNichol’slabhttp://cimed.wustl.edu/

additionalcomplexities•  Phenotypemaydependongeneticbackground•  sideeffectsoftherapeuticdrugsmayinvolveacuteorchronicchangesinionchannelsortransporters(kindlingeffect)

•  Autoantibodies–myastheniagravis,anti-NMDAreceptorencephalitis

AdditionalReading•  HodgkinAL,HuxleyAF.(1952)Aquantitativedescriptionofmembrane

currentanditsapplicationtoconductionandexcitationinnerve.JPhysiol.117:500-44.

•  Neher E, Steinbach JH. (1978) Local anaesthetics transiently block currents through single acetylcholine-receptor channels. J Physiol. 277:153-76.

•  HoshiT,ZagottaWN,AldrichRW.(1990)BiophysicalandmolecularmechanismsofShakerpotassiumchannelinactivation.Science.250:533-8.

•  ZagottaWN,HoshiT,AldrichRW.(1990)RestorationofinactivationinmutantsofShakerpotassiumchannelsbyapeptidederivedfromShB.Science.250:568-71.

•  AshcroftFM.(2006)Frommoleculetomalady.Nature.440:440-7.

•  KimJB.(2014)Channelopathies.KoreanJPediatrics.57:1-18.