CELLULAR CARDIAC

ELECTROPHYSIOLOGICAL TECHNIQUES

NORBERT JOST, PhD

Electrical model of the membrane

Standard intracellular microelectrode technique

Voltage clamp technique

Patch clamp technique

G=1/R

Ohm’s law Ion channel model

Current clamp

Voltage clamp

Intracellular microelectrode technique

Re << Rin

Rin = 1012 Ohm

0.1 - 0.2 m

Ag/AgCl3 M KCl

Re ~ 10 - 40 MOhm

The setup

Organ bath

d: stimulating electrode

e: microelectrode

r: referent electrode

P: preparation

computer

A/D

ingerlő

amplifier

Detected signalP

de

r

100 ms

50 m

V

0mV

drug

60 min 20-60 min 60 min

0 mV

20 mV

100 msAPA

RPAPD50

APD90

90%

50%

Vmax

Wash-outPre-incubation

Two microelectrode voltage clamp

voltage command

holding potential

test potential

The macroscopic sodium current

The voltage-clamp circuit

voltage command

amplifier

Current measure

voltage measure

follow upamplifier

Patch-clamp: the special case of the voltage clamp

Cell

Patch-clamp: the special case of the voltage clamp

(1) Suck a small piece of membrane onto the tip of a glass micropipette (~ 1 µm in diameter)

Cell

(2) “Gigaohm-seal”

R > 1 GOhm

Patch-clamp: the special case of the voltage clamp

Cell

(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.

Patch-clamp: the special case of the voltage clamp

closed

open

Cell+ +

Patch-clamp: the special case of the voltage clamp

(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.

closed open

open

Cell

(3) Turn on the aimed potential the inside part of the pipette and keep it constantly by applying the voltage clamp technique.

Patch-clamp: the special case of the voltage clamp

voltage command

10 msec

Properties of individual voltage-dependent sodium channels

1. Individual channels are either open or closed (no partial openings)

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

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.

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

Properties of individual voltage-dependent sodium channels

The macroscopic sodium current

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.

Properties of individual voltage-dependent sodium channels

The macroscopic sodium current

Summation of 300 recordings

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.

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.

Summation of 300 recordings

Properties of individual voltage-dependent sodium channels

The macroscopic sodium current

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.

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)

Summation of 300 recordings

Properties of individual voltage-dependent sodium channels

The macroscopic sodium current

Slowly inactivating K current channel (Ram & Dagan, 1987)

1. Individual channels are either open or closed (no partial openings). Sometimes more than one channel is in a patch.

2. Each channel opening is only a brief event compared to the total duration of the whole 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.

Similarly, individual potassium channels, calcium channels, and other channels

can be studied by patch clamping

On-Cell

Cell-Attached

The configurations of the patch-clamp technique

On-Cell

Inside-out patch

The configurations of the patch-clamp technique

Whole Cell

On-Cell

The configurations of the patch-clamp technique

Whole Cell

The configurations of the patch-clamp technique

Whole Cell

outside-out patch

The configurations of the patch-clamp technique

Rs

CmRc

The whole-cell configuration

NaCl 144NaH2PO4 0.4

KCl 4

MgSO4 0.53

CaCl2 1.8

Glucose 5.5

HEPES 5

+

ICa blocker

Intracellukar 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

The whole cell configuration

The “run-down“ effect

The ATP-sensitive potassium current

The L-type calcium current

The “run-down“

Whole Cell

Whole Cell, perforated patch

- amphotericin-B- nystatin

The configurations of the patch clamp technique

The “run-down”

The L-type calcium current

Cell isolation

- Ca2+ - free perfusion

- enzymatic digestion (collagenase)

- mechanical separation

200 pA

100 ms

0 mV400 ms

-40 mV

L- type calcium current (ICa)

-40 -20 0 20 40 60

-1200

-800

-400

0

I Ca

amp

lítú

dó

(p

A)

Potenciál (mV)

-35 mV-40 mV

L- type calcium current (ICa)

-40 -20 0 20 40 60

-1200

-800

-400

0

I Ca

amp

lítú

dó

(p

A)

Potenciál (mV)

-30 mV-40 mV

L- type calcium current (ICa)

-40 -20 0 20 40 60

-1200

-800

-400

0

I Ca

amp

lítú

dó

(p

A)

Potenciál (mV)

-25 mV-40 mV

L- type calcium current (ICa)

-40 -20 0 20 40 60

-1200

-800

-400

0

I Ca

amp

lítú

dó

(p

A)

Potenciál (mV)

-20 mV-40 mV

L- type calcium current (ICa)

-40 -20 0 20 40 60-1200

-800

-400

0

I Ca

amp

lítú

dó

(p

A)

Potenciál (mV)

-15 mV-40 mV

L- type calcium current (ICa)

-40 -20 0 20 40 60-1200

-800

-400

0

I Ca

amp

lítú

dó

(p

A)

Potenciál (mV)

-10 mV-40 mV

L- type calcium current (ICa)

-40 -20 0 20 40 60-1200

-800

-400

0

I Ca

amp

lítú

dó

(p

A)

Potenciál (mV)

55 mV

-40 mV

drug

5-10 min 3-5 min 10-15 min

Wash-outPre-incubation

Current-voltage (I-V) relationship

L- type calcium current (ICa)