心臟電生理技術(shù)基礎(chǔ)(膜片鉗)

CELLULARCARDIACELECTROPHYSIOLOGICALTECHNIQUESNORBERT

JOST,PhDElectricalmodelofthe

membraneStandard

intracellularmicroelectrodetechniqueVoltageclamptechniquePatchclamptechniqueG=1/RGtotal

=2Glipid

bilayerio

nchannelG

total=2YG△V=IR=I/G(units:volts)channel

model.10工

之Ohm'slawlonEreversal△V=IR[G=I/RYVoltage

clampCurrent

clampCapacitanceConductanceCIntracellular

microelectrode

techniqueRe<<RinRin

=1012ABEQUIVALENT

CIRCUITOhmsilver

wireelectron(e-)flowAgCl

CoatingAElectrodereaction:Ag+Cl-=AgCl+electron(e-)Thisreactioncanalsobepresentedby:copper

wirecgA0.1

-0.2

μmAg/AgCl

3

MKCIRe~10-40MOhm2.0

mmheatingglass

tubingQO

OcableStop二

二

二-solenoidcoilpipette

carrierfilamentplunger-

solenoidstopQA/DeDetected

signald:stimulatinge:

microelectroder:

referentP:preparationTheamplifiefelectrodeelectrodeingerlódPsetupcomputerOrgan

bath

0mV100

ms50

mVPre-incubation

drug

Wash-out60

min

20-60

min

60

mintest

potentialvoltage

commandholdingpotentialTwomicroelectrodevoltageclampThe

macroscopicsodiumcurrentThe

voltage-clampcircuitUki4-I'amplifierTLU。voltagecommandV

=0mVm=VmImers=0CurrentmeasurefollowamplifjervoltagemeasureRs,uUmRsImem×Iup十Patch-clamp:thespecialcaseofthe

voltageclampPatch-clamp:thespecialcaseofthe

voltageclamp(1)Sucka

small

~i

m

r

neteμma1piece

oftmh

mti

r

lmicropipetteassntogoaeoapbeeCellPatch-clamp:thespecialcaseofthe

voltageclampCellPatch-clamp:thespecialcaseofthe

voltageclamp(3)Sensevoltage

here,inside

theelectrode,anduse

voltageclamp

to

keepit

constant.CellPatch-clamp:thespecialcaseofthe

voltageclamp(3)Sensevoltage

here,

o

ande,edthrectideleinsclosedOpenuse

voltageclamp

to

keepit

constant.十CellPatch-clamp:thespecialcaseofthe

voltageclampclosedOpen(3)Turn

ontheaimed

potentialthe

inside

part

ofthe

pipette

andkeep

it

constantlyby

applying

thevoltage

clamptechnique.CellOpenPropertiesofindividualvoltage-dependentsodiumchannels

voltagecommand10

msecuy—

———v

Propertiesofindividualvoltage-

dependentsodiumchannels1.

Individualchannelsareeitheropenorclosed(no

partialopenings)

~

—————v————~—sodiumcurrentPropertiesofindividualvoltage-

dependentsodiumchannels1.

Individual

channels

are

either

openor

closed

(no

partial

openings)2.

Each

channelopening

isonly

a

briefevent

compared

to

the

total

durationof

the

whole

cell

voltage-dependentsodium

current.Propertiesofindividualvoltage-

dependentsodiumchannels1.

Individual

channels

are

either

open

or

closed

(no

partial

openings)2.

Each

channel

opening

is

only

a

brief—

r

———

event

compared

to

the

total

duration

of

the

whole

cell

voltage-dependent

一

廠

—

—3.

lc

g

and

closing

is

Jvnt.ninereprounemnuChsovariable

in

duration

and

latency.

~

TPropertiesofindividualvoltage-

dependentsodiumchannelsSummationof300recordings

1.

The

channels

are

either

in

open

orclosed

state.2.

The

channel

openings

are

shortevents

when

compared

with

themacroscopic

sodium

current.3.

The

time

duration

and

latency

of

thechannel

openings

are

variable(casesensitive).Might

happen

to

not

openat

all.4.The

open

probability

of

the

channelsresembles

with

that

of

theThemacroscopic

sodiumcurrentmacroscopic

current.Propertiesofindividualvoltage-

dependentsodiumchannels1.

I

u

oc

l

i

r

open

orshgitnenepearosaletinrnaaph(nalddseivicln2.

Each

channel

opening

is

only

a

briefevent

compared

to

the

total

durationof

the

whole

cell

voltage-dependent3.

lc

g

and

closing

isopeninurrent.nneumChsovariable

in

duration

and

latency.4.Theoverallprobability

of

channelopening

is

similar

to

the

total

sodiumcurrent.Look

at

the

sum

of

thecurrents

from

300

trials.5.

Sometimes

an

individual

channelTdoesn't

open

even

once.The

macroscopic

sodiumcurrentSummationof300recordings—Propertiesofindividualvoltage-

dependentsodiumchannels1.

loc

l

i

er

open

or2.

Eachchannelopening

is

only

a

briefs)thngeiopens

arepartialhannenasedividclIneventcomparedto

the

total

durationof

the

whole

cell

voltage-dependent3.

g

and

closing

isnnel

openinum

current.Chsovariable

in

duration

and

latency.4.The

overall

probability

of

channelopening

is

similar

to

the

total

sodiumcurrent.Look

at

the

sum

of

thecurrents

from300

trials.5.

sope

neve

d

.

channelceualnidoivninna'tenmstidoeSome——T6.

Second

openings

are

rare

(becauseThemacroscopic

sodiumcurrentSummationof300recordingsof

inactivation)1.

u

oc

l

i

r

open

orSometimes

more

than

one

channel

is5.

Second

openings

can

happen

if

there's

no

inactivation.shgitnenepearosialnertnaaph(nalddseiviclInSimilarly,individualpotassiumchannels,calciumchannels,andotherchannelscanbestudied

by

patchclampingSlowly

inactivating

R

re&nDt

el1987),naganchanmuracin

a

patch.2.

Eachchannelopening

is

only

a

briefevent

compared

to

the

total

durationof

the

whole

cell

current.3.

Channel

opening

and

closing

isvariable

in

duration

and

latency.4.The

overallprobabilityofchannelopening

is

similar

to

the

whole

cellcurrentTheconfigurationsofthepatch-clamptechniqueCell-AttachedTheconfigurationsofthepatch-clamptechniqueInside-out

patchThe

configurations

ofthe

patch-clamp

techniqueTheconfigurationsofthepatch-clamptechniqueTheconfigurationsofthepatch-clamptechniqueoutside-out

patchcontainsIntracellularsolutionWhole-cell

recording

andoutside-out

patchesForma

gigasealPatchExtracelular

solutionCell-attached

and

inside-out

patchesCell

attachedPullOutside-outpatchApplystrongsuction

to

achieve

whole-cellrecordingmodeInside-outpatchpipcttePullPullThe

whole-cell

configurationip=im+icVp-Vc=ipRsVp

Ve(o)t=Cm.RsRc/(Rs+Rc)VeipExtracellularsolution(mM)NaCI

144NaH?

PO?0.4KCI4MgSO?0.53CaCl?1.8Glucose

5.5HEPES

5十Ica

blockerIntracellukarsolution(mM)K-aspartate

100

KCI

25K?

HPO?

10,K?

EGTA

5K?ATP

3MgCl?1HEPES

10(forK

currents)(forK

currents)ThewholecellconfigurationPatch-clampamplifier昌BM

P

C-20

mV…+50

mV-40

mVExtracellular

solutionIntracellular

solutionMicropipette10

ms

..5000

ms:

:一:

-

一·

一

·-

—:

?

一:一—

:

-

·

—

·

—-

—

-—十一

一—

———-—

-—-

----Figure

87.The

tip

ofa

patch

pipette

formed

from

thin-walled

soft

glass

as

describedby

Hamilletal.(1981).(A)End-on

view

bySEM.(B)Sideviewof

thesamepipetteby

SEM.Scale

bars

represent1.0μm

in(A)and

4.0μm

in(B)(C)Reconstructionof

longitudinal

section

of

this

pipette,which

had

a

cone

angle(φ)of24(From

Sak-mann

andNcher(1983b),reproducedbypermission

ofPlenum

Publishing

Corporation,New

York,N.Y.)0

mM

ATP(0

uM

Ca2+)→5

min1

minTheATP-sensitivepotassiumcurrent0.3

mMATPThe'run-down'effect10pAThe“run-down“The

L-typecalciumcurrentNomalized

le"NTime/minTheconfigurationsofthepatchclamptechniq