single channel analysis and channelopathies...ion channel and transporter pathologies diseases or...
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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.
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