Patch clamp technique

Patch-clamp

techniqueHOVSEP GHAZARYAN

Introduction

The patch clamp technique is a laboratory

technique in electrophysiology that allows the

study of single or multiple ion channels in cells.

The patch clamp technique is a refinement of

the voltage clamp. Erwin Neher and Bert

Sakmann developed the patch clamp in the

late 1970s and early 1980s.

Neher and Sakmann received the Nobel Prize in

Physiology or Medicine in 1991 for this work.

Applications

Neurons

Cardiomyocytes

Muscle fibers

Panceratic betta cells

Bacteria

Kidney cells

Applications

For evaluation of antiarrythmicsagents

To study a cardio selective inhibition of ATP sensitive potassium channel

To identify multiple types of calcium channels

Applications

To measure the effect of potassium channel openers

Voltage clamp studies on sodium channels

To investigate wide range of electropysiological cell properties

Measurement of cell membrane conductance

Historical development

Swammerdam

• Earliest experiments in electrophysiology

Galvani

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

Hodgkin & Huxley

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

Historical development

Graham

• Impaling micropipettes developed by skeletal muscle fibers

Cole & Marmont

•Voltage clamp combined with micropipettes

Sakmann & Neher

•The patch-clamp technique

Need of patch-clamp

Patch-clamp is refinement of voltage clamp technique

Provides for low-noise recordings of currents

Provides access to the inside of cell

•Can insert an electrode into the cell

•Can change intracellular fluid

Creates a seal impermeable to ion flow

•High electrical resistance

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

The patch-clamp technique

Basic principle

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 wit suitable electrolyte solution,

against the surface of a cell and applying light suction

10GΩ resistor at 20C, the standard deviation of the

current noise at 1kHz will be 0.04pA

The patch-clamp circuit

The patch-clamp circuit

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 50GΩ allowing very small

currents (10-12A) to be measured

Cell

Patch-clamp

Suck a small piece of

membrane onto the

tip of a glass

micropipette

(~ 1 µm in diameter)

Cell

“Gigaohm-seal”

R > 1 GOhm

Patch-clamp

Cell

Sense voltage

here, inside the

electrode, and

use voltage clamp

to keep it

constant.

Patch-clamp

closed

open

Cell+ +

Patch-clamp

Sense voltage

here, inside the

electrode, and

use voltage clamp

to keep it

constant.

closed closed

open

Cell

Turn on the aimed

potential the inside

part of the pipette

and keep it

constantly by

applying the voltage

clamp technique.

Patch-clamp

Recording

Recording

Many patch clamp amplifiers do not use

true voltage clamp circuitry, but instead are

differential amplifiers that use the bath

electrode to set the zero current (ground) level.

Current is then injected into the system to

maintain a constant, set voltage.

Alternatively, the cell can be current clamped in

whole-cell mode, keeping current constant

while observing changes in membrane voltage.

voltage command

10 msec

Properties of individual voltage-

dependent sodium channels

1. Individual channels are either open or closed

(no partial openings)





2. Each channel opening is only a brief event

compared to the total duration of the whole

cell voltage-dependent sodium current.

The macroscopic sodium current








3. Channel opening and closing is variable in duration and latency.




1. The channels are either in open or closed state.

2. The channel openings are short events when

compared with the macroscopic sodium

current.

3. The time duration and latency of the channel

openings are variable (case sensitive). Might happen to not open at all.

4. The open probability of the channels

resembles with that of the macroscopic

current.




Summation of 300 recordings






3. Channel opening and closing is variable in duration and latency.

4. The overall probability of channel opening is

similar to the total sodium current. Look at the

sum of the currents from 300 trials.

5. Sometimes an individual channel doesn’t open

even once.




The macroscopic sodium

current






3. Channel opening and closing is variable in

duration and latency.4. The overall probability of channel opening is

similar to the total sodium current. Look at the

sum of the currents from 300 trials.

5. Sometimes an individual channel doesn’t open

even once.

6. Second openings are rare (because of inactivation)




The macroscopic sodium

current

Slowly inactivating K current

channel

(Ram & Dagan, 1987)


(no partial openings). Sometimes more than

one channel is in a patch.



cell current.3. Channel opening and closing is variable in

duration and latency.

4. The overall probability of channel opening is

similar to the whole cell current

5. Second openings can happen if there’s no

inactivation.

Other channels

Variations of patch-clamp

Cell-attached patch

Inside-out patch

Whole-cell recording or whole-cell patch

Outside-out patch

Perforated patch

Loose patch

Cell-attached patch

Cell-attached patch

Allows the recording of currents through single, or a few, ion

channels contained in the patch of membrane captured by the

pipette. By not disrupting the interior of the cell, any intracellular mechanisms normally influencing the channel will still be able to

function as they would physiologically.

The technique is thus limited to one point in a dose response

curve per patch.

Voltage-gated ion channels can be clamped successively at

different membrane potentials in a single patch.

Inside-out patch

Inside-out patch

The experimenter has access to

the intracellular surface of the

membrane via the bath and can

change the chemical

composition of what the surface

of the membrane is exposed to.

Whole-cell patch

Whole-cell patch

Larger opening at the tip of the patch clamp

electrode provides lower resistance and thus

better electrical access to the inside of the cell.

Because the volume of the electrode is larger

than the volume of the cell, the soluble contents

of the cell's interior will slowly be replaced by the

contents of the electrode.

Outside-out patch

Outside-out patch

Complementarity to the inside-out technique.

Places the external rather than intracellular

surface of the cell membrane on the outside of

the patch of membrane, in relation to the patch

electrode.

The longer formation process involves more steps

that could fail and results in a lower frequency of

usable patches.

Perforated patch

Perforated patch

Similar to the whole-cell configuration

Suction is not used to rupture the patch membrane

The electrode solution contains small amounts of an

antifungal or antibiotic agent, which diffuses into the

membrane patch and forms small pores in the

membrane

The perforated patch can be likened to a screen door

that only allows the exchange of certain molecules from

the pipette solution to the cytoplasm of the cell

Loose patch

Loose patch

Employs a loose seal (low electrical resistance) rather

than the tight gigaseal used in the conventional

technique.

The pipette is moved slowly towards the cell, until the

electrical resistance of the contact between the cell.

and the pipette increases to a few times greater

resistance than that of the electrode alone.

The pipette that is used can be repeatedly removed

from the membrane after recording, and the membrane

will remain intact.

Rs

CmRc

Whole-cell configuration

NaCl 144

NaH2PO4 0.4

KCl 4

MgSO4 0.53

CaCl2 1.8

Glucose 5.5

HEPES 5+

ICa blocker

Intracellular solution (mM)

(for K currents)

Extracellular solution (mM)

(for K currents)

K-aspartate 100

KCl 25

K2HPO4 10

K2EGTA 5

K2ATP 3

MgCl2 1

HEPES 10

Extracellular

solution

Patch-clamp amplifier

IBM PC

Micropipette

+ __

++

+ +

+_

_

__ _

++

_

_

++_

Cell

-40 mV

-20 mV ... +50 mV10 ms ... 5000 ms

Intracellular

solution

Whole cell configuration

Patch-clamp technique in isolated

cardiac myocytes

Perfusion of section of intact

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

37C

Male wistar ratIntact heart Section

Wedge preparation

Perfusion (Physiologic saline → Ca2+

free saline → Ca2+ free enzyme)Dissection

Epicardial preparation Batch digestion Filtration

Isolated myocytes Incubation buffer (0.5M Ca2+

→ 1M Ca2+ )

Electraphysiologic study

Principle & procedure

The generation of an action potential in hearth

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 experiments

Glass micro-pipette - a tip opening of about 1

µm, is placed onto the cell


The patch-pipette is filled with either 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 electrical

amplifier

A second chlorided silver wire is inserted into the

bath and serves a ground electrode

Whole cell patch clamping is done


High input resistance enables the recording of

small electrical currents, which are flowing

through channel forming proteins in the

membrane patch

The electrical current is driven by applying an

electrical potential across membrane patch,

and/or by establishing an appropriated

chemical gradient for the respective ion species


It is important 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)

Evaluation

Concentration-response curves of drugs which

either inhibit or activate ion channels can be

recorded either on he single channel level or by

measuring the whole-cell current

IC50 and EC50 values (50% inhibition or

activation, respectively) can be obtained

Limitations

Requires strong background in ion channel

biophysics

Imparting skillful training performance during

single channel recordings

Cost of process is expensive

Time consuming

Number of samples required is more at times

Chance of membrane distortion

Conclusion

Patch-clamp is highly modified and successful technique

Development of this technique is being done for newer approaches to yield accurate and efficient information which aids drug discovery process.

Thank you

Science

Patch clamp technique