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Transcript of EECE432 2011 Neural
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NeuralapplicationsofMEMS.
invitrorecordingfromneuronsortissue membranestructureandpropertiesofexcitablecells
ionchannels
inchannelstructure restin otentialofmembrane
membraneelectricalmodel
patchclamp convent ona g assp pette
microfabricatedplanarpatchclamp
2. Surfacemodification molecularpatterning
controlattachmentofneuronsonelectrodes
3. Implantablemicroelectrodesandmicroneedles
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invivorecordingfromneuraltissue1
NeuralapplicationsofMEMS(1)
Attheendofthissectionthestudentshouldbeableto:
describeionchannelstructureandfunction,andwhyionchannelsaretargetsfordrugresearch
describetheelectricalmodelofthemembrane
relevantions
describetheconventionalpatchclamptechnique listreasonsdrivingthedevelopmentofhigherthroughput
patchclamptechniques
describeplanarpatchclamparrays
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Furtherreading JOHNSTON D. & WU S. M.S. 1994 Foundations o Cellular
Neurophysiology,MITPress.
, . ., , . . , . .PrinciplesofNeuralScience,McGrawHillMedical.
PURVES,D.,AUGUSTINE,G.J.,FITZPATRICK,D.,HALL,W.C.,LAMANTIA,A.S.,MCNAMARA,J.O.&WILLIAMS,S.M.(Eds.)2004 Neuroscience Sinauer Associates.
http://www.ncbi.nlm.nih.gov/books/NBK26910/
IonChannelsandtheElectricalPropertiesofMembranes
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, .andBioelectricInterfaces
3
Excitablecells
Cellsthatcangenerateelectricalpotentials
Thesepotentialsandcurrentscanbeobservedin
,
membrane,andinthesurroundingconducting
volume.nervecells(neurons)
musclefibers
sensoryreceptor(transducer)cells
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Neurons
EECE432 2011 KCheung 5http://www.ncbi.nlm.nih.gov/books/NBK26910/figure/A2040/
Sensoryreceptorcells
EECE432 2011 KCheungMolecular Biology of the Cell, Alberts et al. 6
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Bioelectricalsystems
chargecarriers:electrons
withinaconductor
chargecarriers:ionswithin
anelectrolyte
currentflowwithin currentflowinsideand
insulated)cellmembranes
insulator cellmembrane
electrolyteNa+,K+, Cl,Ca2+
econductor
Na+,K+, Cl,Ca2+electrolyte
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Typicalionconcentrations
EECE432 2011 KCheungFoundations of Cellular Neurophysiology, Johnston and Wu 8
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Transportproteins ,
passive transportthrough
e ec roc em ca gra en
chemicalgradient
active transport(=needsenergy)
+ +
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Ionchannels integralmembraneprotein(canbeassembledbyseveralsubunitsorbyasingle
su un t
spantheentiremembrane
whenopen,allowfreediffusionofions
Gatin ofthechannelcanbeactivatedb volta eand/ors ecificli andormembranestretch.
Voltagedependentgatedchannelsareattheoriginofthespecificityofthenervouscellsandallowgenerationandpropagationofthenerveimpulse.
Li and atedchannels la afundamentalroleinsi nal chemical transmissionandamplificationbetweencells.
EECE432 2011 KCheungfigure from Neuroscience, Purves et al 10
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Ionchannels
allowionstopassthroughthemembraneunderthe
andelectricpotential
gradients
v yonlyallowcertainionstopassthrough
ionchannelgatesregulatethepermeabilityofthechannels,allowingcontrolovertheflowofions
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Ionchannelstructure
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Functionsofionchannels
knownbiologicalfunction regulationofmembranepotential si naltransduction insulinsecretion hormonerelease re ulationof
cellvolume,immuneresponse ionchanneldysfunctioncanhaveastrongimpactoncellsignallingand
cancausedisorderssuchascardiacarrhythmia. robust assa s stems for characterization
canscreenthousandsofcompoundsagainstionchannels selectionoftargetsthatonlyactontheintendedtarget lesslikelyto
havesideeffects
y y u weightcompounds
genesequencinghasshownthereare>300ionchannelenes
targetforsmallmoleculedrugs
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Tetrodotoxin,isolatedfrompufferfish,bindstightly
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an speci ica ytoNa+ c anne sinnervece s.As itt e
as10ng isfataltohumans.
14http://www.ncbi.nlm.nih.gov/books/NBK22509/#A1818
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Foxg ove Digita ispurpurea ist esourceo igita is,oneofthemostwidelyuseddrugs.Digitalisincreases
theforceofcontractionofheartmuscleandisused
totreatcon estiveheartfailure.
DigitalisinhibitstheNa+ K+ pump,leadingtohigher
[Na+]insidethecell.Thediminishedgradientresults
inslowerextrusionofCa2+ bytheexchanger.Higher
EECE432 2011 KCheung
n race u ar a en ances ea yo car ac
muscletocontract.
15http://www.ncbi.nlm.nih.gov/books/NBK22464/#A1791
Ionsflowthroughopenchannels in excitable cells the une ual concentration of ions
intheintracellularandextracellularspacesproducesionflowthroughanyopenchannels.
ionsaccumulateonthemembrane,producinganelectricfieldacrossandwithinthemembranethatwillexertaforceonchargedparticleswithinionchannels.
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Ionfluxacrossthemembrane
A.InacellpermeableonlytoK+ the
restingpotentialisgeneratedbythe
B.Thebuildupofchargeactstoimpede
furthereffluxofK+,sothateventuallyan
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e uxo own sconcen ra on
gradient.
equ r um sreac e c em ca an
electricaldrivingforcesareequaland
opposite).Principles of Neural Science, Kandel et al. 17
Ioniccompositionandmembranepotential Formostcells,sodium(Na+)andpotassium(K+)arethemostimportantionsfor
theelectricalactivity. calcium(Ca2+)andchloride(Cl)aresignificantinsomecells.
ANernstequilibriumisachievedforaparticularionwhenthe electric field forcebalancestheforceoftheconcentrationgradientforthation,
throughanionchanneliszero.
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Nernstpotential
ThemembranepotentialatwhichK+ ionsareinequilibriumacrossthemembrane.
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Nernstpotential
TheNernstpotentialforaparticularionisreferredtoastheequilibriumpotential.
TheequilibriumpotentialforsodiumionsisENa,theu u u K.
Thee uilibrium otentialisalsocalledthereversalpotential.
,(frominwardstooutwardsorviceversa).
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Membranecapacitance
resistanceof109 /cm2 (insulator)
chargecanbuilduponeachsideofthemembraneinregionswheretherearenoc anne sorw erec anne sarec ose .
mem ranecapac tance m~ . cm .
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Electricalmodelofthemembrane
EECE432 2011 KCheungPrinciples of Neural Science, Kandel et al. 22
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Electricalmodelofthemembrane
passiveproperties
o g n ux eymo e
EECE432 2011 KCheungPrinciples of Neural Science, Kandel et al. 23
Ioniccurrents
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Restingmembranepotential
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Membraneconductance/resistanceatrest ,
restingmembraneconductanceG(totalresting
membraneresistanceR)canbedeterminedfromthe
restingvaluesoftheionicconductances.
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Patchclamp Thepatchclamptechniqueisamethodthatallowsdirectmeasurementof
currentsthroughionchannelswithsubmillisecondresolutionusingglassmicropipettessealedagainstthecellmembrane.
membraneandtheapplicationofsignallingmolecules,drugs,etc.,tobothsides
ofthemembrane.
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Patchclamp
Principles of Neural Science, Kandel et al.
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Channelgating
Ligandgatedchannelsopeninresponseto
bindingoftheligandtoitsreceptor.
Voltagegatedchannelscaneitheropenorclose
inresponsetochangesinmembranepotential.
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Otherchannelsareactivatedbystretchor
pressure.Principles of Neural Science, Kandel et al. 29
Bindingofexogenousligandsreversible anta onist
irreversible
antagonist
endogenous
agonist
Example:thechannelis
normallyopenedbythe
bindingofanendogenousgan .
Adrugortoxinmayblockthe
bindingoftheactivatorby
eitherreversibleorirreversible
reaction.
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Gramicidin to ical antibiotic causes hemol sis at low concentrations
cannotbeadministeredinternally)
increasesthepermeabilityofthebacterialcellwallby
destroystheiongradientbetweenthecytoplasmandthe
extracellularenvironment
EECE432 2011 KCheungPrinciples of Neural Science, Kandel et al. 31
Gramicidin
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currentthroughchannelchangesproportionallywiththeelectricaldrivingforce
Principles of Neural Science, Kandel et al. 32
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Recordingcurrentflowfromsingleionchannels
Ametalelectrodeincontactwiththe
electrolyteinthemicropipetteconnectsit
toanelectricalcircuitthatmeasuresthe
currentthatflowsthroughchannelsinthe
EECE432 2011 KCheungPrinciples of Neural Science, Kandel et al.
.
33
Patchclamp
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synapticallyconnectedlayer5pyramidalneuronsina
corticalbrainslice.
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Chipbasedplanarpatchclamp
withaplanararrayofrecordinginterfaces
miniaturizedonthesurfaceofeitherasilicon,
polymerorglasssubstrate.
traditionalpatchclamp:highqualitydata,butlow
throughputandhighuserskill.
ncrease roug pu s mu aneousrecor ngs
ecreasecostper atapoint
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Siliconnitridemembrane ositionin vesiclesonan
aperture(diameter~0.67m)
thicknessofsiliconnitridemembraneh~100nm
surfaceofnitridemembranemodifiedtoattractthe
negativelychargedvesicles
measuredionchannelcurrents
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SCHMIDT, C., MAYER, M. & VOGEL, H. (2000) A Chip-Based Biosensor for the
Functional Analysis of Single Ion Channels.Angewandte Chemie, 39, 3137-3140. 36
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PDMSbasedplanarpatchclamp
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LI, X., KLEMIC, K. G., REED, M. A. & SIGWORTH, F. J. (2006) Microfluidic System for
Planar Patch Clamp Electrode Arrays. Nano Letters, 6, 815-819. 37
EECE432 2011 KCheung
LI, X., KLEMIC, K. G., REED, M. A. & SIGWORTH, F. J. (2006) Microfluidic System for
Planar Patch Clamp Electrode Arrays. Nano Letters, 6, 815-819. 38
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Glassbasedplanarpatchclamp
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FERTIG, N., BLICK, R. H. & BEHRENDS, J. C. (2002) Whole Cell Patch Clamp
Recording Performed on a Planar Glass Chip. Biophysical Journal, 82, 3056-3062.
Nanion Technologies GmbH
39
Siliconbased
EECE432 2011 KCheungCytocentrics, Germany 40
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AutomatedElectrophysiology
1038/nrd2552
2008)|
doi:10.
,358
368(April
DrugDiscovery
ature
Reviews
Manualelectrophysiology 2
:10.1
038/nrd25
pril2008)|doi
ry7,
358
368(
wsDrugDiscov
NatureRevie
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