PATCH CLAMP TECHNIQUE

BY- RITIK VARDHAN

M.Sc.(P)
ROLL NO.- 1366

CONTENTS

INTRODUCTION
HISTORY
PRINCIPLE
CONFIGURATION & TYPES
APPLICATION
LIMITATION
RECENT RESEARCH

INTRODUCTION

Thepatchclamptechniqueisalaboratorytechniquein
electrophysiologythatallowsthestudyofsingleor
multipleionchannelsincells.
SakmannandNeher-developthepatchclamptechnique
in1970sandearly1980s.
ReceivedtheNobelprizeforthishighscientificwork
in1991.

HISTORICAL

DEVELOPMENT

Jan
Swammerda
m

Luigi
Galvani

earliest
experiment
sin
electrophys
iology

thefirst
experiment
alevidence
ofelectrical
activityin
animalsby
usingmetal
wiresin
frogmuscle

Hodgkinand
Huxley

thefirst
intracellula
r
measureme
ntofthe
action
potentialin
thegiant
squidaxon

Graham

Impaling
micropipett
es
developed
byskeletal
muscle
fibres

ColeandMarmont

Voltageclamp
technique
combinedwith
micropipettes

Sakmannand
Neher

thepatchclamp
technique

NEED

OF PATCH CLAMP

Patchclampisrefinementofvoltageclamptechnique.
providesforlow-noiserecordingsofcurrent
Providesaccesstotheinsideofthecell
Caninsertanelectrodeintothecell
Canchangetheintracellularfluid
Createsasealimpermeabletoionflow
Highelectricalresistance
Allowsonetomeasurecurrentthroughionchannelsvs.
voltage,time,temperature.

THE PATCH-CLAMP TECHNIQUE

Erwin Neher
Bert Sakmann
Germany
(1991 Nobel Laureates)

BASICPRINCIPLE
The principle of the method is to isolate a patch of membrane
electrically from the external solution and to record current owing
into the patch
This is achieved by pressing a re-polished glass pipette,
which has been lled with a suitable electrolyte solution,
against the surface of a cell and applying light suction

Electrode (10-25 m)

re -polished glass pipette

Electrolyte solution

7/7/2011

<10nm

10 G resistor at 20C, the standard

8
deviation of the current noise at 1 kHz will
8
be 0.04 pA,

The patch-clamp circuit

FBR
_
+

Amplifier

Technical
The high gain operational amplifier is
connected in the circuit so that the current
flowing through the ion channel is measured
as a voltage drop across the feedback resistor
(FBR). The FBR has a resistance of 50 G
allowing very small currents (10-12 A)
to be measured.

Patch of cell membrane with ion channel

A patch-clamp rig

CONFIGURATION OF PATCH CLAMP:

On-cell

Inside Out

Whole Cell

OutsideOut

11

TYPES

Perforated patch
Loose patch

DPerforated-patch method
(simplified)ASF

Patch clamp technique in isolated

cardiac myocytes

Perfusion of a section of intact canine left ventricular

myocardium. A cannula has been placed into the
left anterior descending coronary artery and clamps
have been placed to occlude major coronary artery
branches that have been transected during sectioning

ISOLATION OF MYOCYTES

Male wistar rat

PRINCIPLE & PROCEDURE

Thegenerationofanactionpotentialinheartmuscle
cellsdependsontheopeningandclosingofionselectivechannelsintheplasmamembrane.
Thepatch-clamptechniqueenablestheinvestigationof
druginteractionswithion-channel.
TheIsolatedcellsarereadyforexperiment.
Glassmicro-pipette-atipopeningofabout1m,is
placedontothecell.

 Thepatch-pipetteisfilledwitheitherhighNaClorKCl
solutionandismountedonamicromanipulator.

Achloridedsilverwireconnectsthepipette

solutiontotheheadstageofanelectronicalamplifier.
Asecondchloridedsilverwireisinsertedintothebathand
servesagroundelectrode.
Wholecellpatchclampingisdone

Thishighinputresistanceenablestherecordingofsmall
electricalcurrentsintherangeofPicosiemens(1012S),

whichareflowingthroughchannel-formingproteinssituated
inthemembranepatch.

Theelectricalcurrentisdrivenbyapplyinganelectrical
potentialacrossthemembranepatch,and/orbyestablishingan
appropriatedchemicalgradientfortherespectiveionspecies.

Toinvestigatetheinteractionofdrugswithallionchannels
involvedinthefunctioningoftheheartmusclecell(K+,Na+,
Ca2+andeventuallyClchannels).

ThedifferenttypesofK+channelsexistingincardiomyocyte.

EVALUATION

Concentration-responsecurvesofdrugswhicheither
inhibitoractivateionchannelscanberecordedeither
onthesinglechannellevelorbymeasuringthewhole
cellcurrent.IC50 and EC50 values (50% inhibition or
activation,respectively)canbeobtained.

limitations

Impartingskillfultrainingperformanceandrecording
Induringsinglechannelrecordings
Costofprocessisexpensive
Timeconsuming
Numberofsamplesrequiredismoreattimes
Chanceofmembranedistortion

APPLICATIONS

Fortheevaluationofantiarrhythmicsagents.
Inkidneycells.

UsedforisolatedventricularmyocytesfromGuineapigstostudyacardio
selectiveinhibitionoftheATPsensitivepotassiumchannel.
Toidentifymultipletypesofcalciumchannels.
Tomeasuretheeffectofpotassiumchannelopeners.
Usedinthemolecularbiology.
Voltageclampstudiesonsodiumchannels.
Usedtoinvestigateawiderangeofelectrophysiologicalcellproperties.
Measurementofcellmembraneconductance.

RECENT RESEARCH

Measurementsareconductedinamultiparametric

mannerinanintegratedandautomatedmicrofluidic
chip.
Micropippetesintraditionalpatchclamptechniqueare
replacedbynanomachinepatchclampsystemwith
integratedmicrofluidicswhichaids
RapidIntracellularperfusion
Improvedopticalmeasurments
Rapidmeasurmentofsinglecelldoseresponse
curves

conclusion

Itishiglymodifiedandsuccessfultechnique
Developmentofthistechniqueisbeingdonefornewer
approachestoyieldbetteraccurateandefficient
informationwhichaidsdrugdiscoveryprocess.

References
1.

2.

Wyllie DJA (2007) Single-channel recording. In Patch-Clamp Analysis

Advanced Techniques 2nd Edition. pp 69-129. Ed. Walz W. Humana Press
Totowa, New Jersey USA

Sakmann B (1992) Elementary steps in synaptic transmission revealed by

currents through single ion channels. Neuron 8, 613-629.
This is his 1992 Nobel Lecture and well worth a read

3.

Aidley DJ & Stanfield PR (1996) Ion Channels Molecules in Action

Cambridge University Press.
This is a very good introductory textbook.