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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

EECE432 2011 KCheung

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


, .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

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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


Typicalionconcentrations

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Transportproteins ,

passive transportthrough

e ec roc em ca gra en

chemicalgradient

active transport(=needsenergy)

+ +


9

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


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


Tetrodotoxin,isolatedfrompufferfish,bindstightly


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


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


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.


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 .


Electricalmodelofthemembrane

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Electricalmodelofthemembrane

passiveproperties

o g n ux eymo e


Ioniccurrents


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Restingmembranepotential


Membraneconductance/resistanceatrest ,

restingmembraneconductanceG(totalresting

membraneresistanceR)canbedeterminedfromthe

restingvaluesoftheionicconductances.


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Patchclamp Thepatchclamptechniqueisamethodthatallowsdirectmeasurementof

currentsthroughionchannelswithsubmillisecondresolutionusingglassmicropipettessealedagainstthecellmembrane.

membraneandtheapplicationofsignallingmolecules,drugs,etc.,tobothsides

ofthemembrane.


Patchclamp

Principles of Neural Science, Kandel et al.


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Channelgating

Ligandgatedchannelsopeninresponseto

bindingoftheligandtoitsreceptor.

Voltagegatedchannelscaneitheropenorclose

inresponsetochangesinmembranepotential.


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


Gramicidin


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


synapticallyconnectedlayer5pyramidalneuronsina

corticalbrainslice.

34

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Chipbasedplanarpatchclamp

withaplanararrayofrecordinginterfaces

miniaturizedonthesurfaceofeitherasilicon,

polymerorglasssubstrate.

traditionalpatchclamp:highqualitydata,butlow

throughputandhighuserskill.

ncrease roug pu s mu aneousrecor ngs

ecreasecostper atapoint


Siliconnitridemembrane ositionin vesiclesonan

aperture(diameter~0.67m)

thicknessofsiliconnitridemembraneh~100nm

surfaceofnitridemembranemodifiedtoattractthe

negativelychargedvesicles

measuredionchannelcurrents


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


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


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


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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