CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES

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CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES

• NORBERT JOST, PhD

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Electrical model of the membrane Standard
intracellular microelectrode technique Voltage
clamp technique Patch clamp technique
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G1/R
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Ohms law
Ion channel model
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Current clamp
Voltage clamp
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Intracellular microelectrode technique
Re ltlt Rin Rin 1012 Ohm
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Ag/AgCl 3 M KCl
Re 10 - 40 MOhm
0.1 - 0.2 ?m
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The setup
amplifier
computer
ingerlo
A/D
d
e
r
Detected signal
P
0mV
Organ bath
d stimulating electrode
50 mV
e microelectrode
r referent electrode
100 ms
P preparation
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0 mV
50
APA
Vmax
90
RP
APD50
APD90
drug
Wash-out
Pre-incubation
60 min
20-60 min
60 min
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Two microelectrode voltage clamp
test potential
voltage command
holding potential
The macroscopic sodium current
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The voltage-clamp circuit
voltage measure
follow up amplifier
voltage command
amplifier
Current measure
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Patch-clamp the special case of the voltage
clamp
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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
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Patch-clamp the special case of the voltage
clamp
(2) Gigaohm-seal
R gt 1 GOhm
Cell
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Patch-clamp the special case of the voltage
clamp
(3) Sense voltage here, inside the electrode, and
use voltage clamp to keep it constant.
Cell
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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

Cell
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Patch-clamp the special case of the voltage
clamp
(3) Turn on the aimed potential the inside part
of the pipette and keep it constantly by applying
the voltage clamp technique.
closed open open
Cell
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Properties of individual voltage-dependent sodium
channels
voltage command
10 msec
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Properties of individual voltage-dependent sodium
channels

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

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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
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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.

The macroscopic sodium current
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Properties of individual voltage-dependent sodium
channels

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
The macroscopic sodium current
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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.
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 doesnt open even
   once.

Summation of 300 recordings
The macroscopic sodium current
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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.
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 doesnt open even
   once.
6. Second openings are rare (because of
   inactivation)

Summation of 300 recordings
The macroscopic sodium current
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Similarly, individual potassium channels, calcium
channels, and other channels can be studied by
patch clamping

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 theres no
   inactivation.

Slowly inactivating K current channel (Ram
Dagan, 1987)
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The configurations of the patch-clamp technique
On-Cell
Cell-Attached
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The configurations of the patch-clamp technique
Inside-out patch
On-Cell
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The configurations of the patch-clamp technique
Whole Cell
On-Cell
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The configurations of the patch-clamp technique
Whole Cell
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The configurations of the patch-clamp technique
outside-out patch
Whole Cell
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The whole-cell configuration
Rs
Cm
Rc
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Intracellukar solution (mM) (for K currents)
Extracellular solution (mM) (for K currents)
NaCl 144 NaH2PO4 0.4 KCl 4 MgSO4 0.53 CaCl2
1.8 Glucose 5.5 HEPES 5 ICa blocker
K-aspartate 100 KCl 25 K2HPO4 10, K2EGTA 5 K2ATP
3 MgCl2 1 HEPES 10
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The whole cell configuration
Intracellular solution
Patch-clamp amplifier
Micropipette
IBM PC

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Extracellular solution
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Cell
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10 ms ... 5000 ms
-20 mV ... 50 mV
-40 mV
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The run-down effect
The ATP-sensitive potassium current
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The run-down
The L-type calcium current
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The configurations of the patch clamp technique
Whole Cell, perforated
patch
Whole Cell
- amphotericin-B - nystatin
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The run-down
The L-type calcium current
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Cell isolation
- Ca2 - free perfusion - enzymatic digestion
(collagenase) - mechanical separation
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L- type calcium current (ICa)
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L- type calcium current (ICa)
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L- type calcium current (ICa)
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L- type calcium current (ICa)
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L- type calcium current (ICa)
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L- type calcium current (ICa)
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L- type calcium current (ICa)
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L- type calcium current (ICa)
Current-voltage (I-V) relationship
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