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PATCH CLAMP TECHNIQUE

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UNDER THE GUIDENCE OF

MISS MEENU SINGH

ASST.PROFEESOR

DEPARTMENT OFPHARMACOLOGY

CMRCP

BY

KRISHNAPRIYA.P

M.PHARM

PHARMACOLOGY

INTRODUCTION

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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HISTORICAL DEVELOPMENT

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Graham

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NEED OF PATCH CLAMP 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 cellCan change the intracellular fluid

Creates a seal impermeable to ion flowHigh electrical resistance

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

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THE PATCH-CLAMP TECHNIQUE

Erwin NeherBert Sakmann

Germany(1991 Nobel Laureates)

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BASIC PRINCIPLE7

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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)

Renitence

Si 2 = 4kTfc /R

variance of the current noise (in A2) k = Boltzmann’s constant, T = temperature (° Kelvin), fc = the bandwidth(Hz)

<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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TYPES OF PATCH CLAMP:

• On-cell

• Inside Out

• Whole Cell

• OutsideOut

TYPES

Perforated patch

Loose patch

Blind patch

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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

ISOLATION OF MYOCYTES

Male wistar rat

PRINCIPLE & PROCEDURE

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.

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

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.

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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EVALUATION

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

wholecell

current. IC50 and EC50 values (50% inhibition or

activation, respectively) can be obtained

LIMITATIONS

Requires strong background inc channel biophysics

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

APPLICATIONS

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.

RECENT RESEARCH

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

Large experimental through put

CONCLUSION

It is higly modified and successful technique

Developmant of this technique is being done for newer

approaches to yield better accurate and efficient

information which aids drug discovery process.

PATCH-CLAMP TCHNIQUE

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