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PATCH CLAMP TECHNIQUE BY- RITIK VARDHAN M.Sc.(P) ROLL NO.- 1366

Patch clamp technique

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Page 1: Patch clamp technique

PATCH CLAMP TECHNIQUE

BY- RITIK VARDHANM.Sc.(P)

ROLL NO.- 1366

Page 2: Patch clamp technique

INTRODUCTION HISTORY PRINCIPLE CONFIGURATION & TYPES APPLICATION LIMITATION RECENT RESEARCH

CONTENTS

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The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in cells.

Sakmann and Neher - develop the patch clamp technique in 1970s and early 1980s.

Received the Nobel prize for this high scientificwork in1991 .

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INTRODUCTION

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Jan Swammerda

m

• earliest experiments in electrophysiology

Luigi Galvani

• the first experimental evidence of electrical activity in animals by using metal wires in frog muscle

Hodgkin and Huxley

• the first intracellular measurement of the action potential in the giant squid axon

• Impaling micropipettes developed by skeletal muscle fibres

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HISTORICAL DEVELOPMENT

Graham

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Cole and Marmont

• Voltage clamp technique combined with micropipettes

Sakmann and

Neher

• the patch clamp technique

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Continues………………….

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Patch clamp is refinement of voltage clamp technique. provides for low-noise recordings of current Provides access to the inside of the cell

Can insert an electrode into the cell Can change the intracellular fluid

Creates a seal impermeable to ion flow High electrical resistance

Allows one to measure current through ion channels vs. voltage, time, temperature.

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NEED OF PATCH CLAMP

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THE PATCH-CLAMP TECHNIQUE

Erwin NeherBert Sakmann

Germany(1991 Nobel Laureates)

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7/7/2011 8

BASIC PRINCIPLE

The principle of the method is to isolate a patch of membrane electrically from the external solution and to record current flowing into the patch

This is achieved by pressing a fire-polished glass pipette, which has been filled with a suitable electrolyte solution, against the surface of a cell and applying light suction

fire -polished glass pipette

Electrolyte solution

Electrode (10-25 µm)

<10nm

10 GΩ resistor at 20°C, the standarddeviation of the current noise at 1 kHz will be 0.04 pA,8

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The patch-clamp circuit

Patch of cell membrane with ion channel

FBR

_

+Amplifier

TechnicalThe high gain operational amplifier isconnected in the circuit so that the currentflowing through the ion channel is measuredas a voltage drop across the feedback resistor(FBR). The FBR has a resistance of 50 Gallowing very small currents (10-12 A)to be measured.

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A patch-clamp rig

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CONFIGURATION OF PATCH CLAMP:

• On-cell

• Inside Out

• Whole Cell

• OutsideOut

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Perforated patch

Loose patch

DPerforated-patch method (simplified)ASF

TYPES

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Patch clamp technique in isolated cardiac myocytes

Perfusion of a section of intact canine left ventricularmyocardium. A cannula has been placed into theleft anterior descending coronary artery and clampshave been placed to occlude major coronary arterybranches that have been transected during sectioning

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Male wistar rat

ISOLATION OF MYOCYTES

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The generation of an action potential in heart muscle

cells depends on the opening and closing of ion-selective channels in the plasma membrane.

The patch-clamp technique enables the investigation of drug interactions with ion-channel .

The Isolated cells are ready for experiment.

Glass micro-pipette - a tip opening of about 1 μm, is

placed onto the cell.

PRINCIPLE & PROCEDURE

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The patch-pipette is filled with either high NaCl or KCl

solution and is mounted on a micro manipulator.

A chlorided silver wire connects the pipette

solution to the head stage of an electronical amplifier.

A second chlorided silver wire is inserted into the bath and

serves a ground electrode.

Whole cell patch clamping is done

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This high input resistance enables the recording of small

electrical currents in the range of Picosiemens (10–12 S),

which are flowing through channel-forming proteins situated

in the membrane patch.

The electrical current is driven by applying an electrical potential

across the membrane patch, and/or by establishing an

appropriated chemical gradient for the respective ion species.

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To investigate the interaction of drugs with all ion channels

involved in the functioning of the heart muscle cell (K+, Na+,

Ca2+ and eventually Cl– channels).

The different types of K+ channels existing in cardiomyocyte.

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Concentration-response curves of drugs which either

inhibit or activate ion channels can be recorded either

on the single channel level or by measuring the whole

cell current. IC50 and EC50 values (50% inhibition or

activation, respectively) can be obtained.

EVALUATION

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Imparting skillful training performance and recording

In during single channel recordings

Cost of process is expensive

Time consuming

Number of samples required is more at times

Chance of membrane distortion

limitations

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For the evaluation of antiarrhythmics agents.

In kidney cells.

Used for isolated ventricular myocytes from Guinea pigs to study a cardio

selective inhibition of the ATP sensitive potassium channel.

To identify multiple types of calcium channels.

To measure the effect of potassium channel openers.

Used in the molecular biology.

Voltage clamp studies on sodium channels.

Used to investigate a wide range of electrophysiological cell properties.

Measurement of cell membrane conductance.

APPLICATIONS

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Measurements are conducted in amultiparametric

manner in an integrated and automated microfluidic chip.

Micropippetes in traditional patch clamp technique are replaced by nano machine patch clamp system with integrated micro fluidics which aids

Rapid Intra cellular perfusion

Improved optical measurments

Rapid measurment of single cell dose response curves

RECENT RESEARCH

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It is higly modified and successful technique

Development of this technique is being done for newer

approaches to yield better accurate and efficient

information which aids drug discovery process.

conclusion

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References1. 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

2. 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.

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