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
Ag/AgCl
3 M KCl
Re ~ 10 - 40 MOhm
0.1 - 0.2 m
The setup
amplifier
computer
ingerlő
A/D
e
P
r
Organ bath
d: stimulating electrode
e: microelectrode
r: referent electrode
P: preparation
Detected signal
0mV
50 mV
d
100 ms
0 mV
20 mV
50%
APA
100 ms
Vmax
90%
RP
APD50
APD90
Pre-incubation
60 min
drug
Wash-out
20-60 min
60 min
Two microelectrode voltage clamp
test potential
voltage command
holding potential
The macroscopic sodium current
The voltage-clamp circuit
follow up
amplifier
amplifier
Current
measure
voltage
measure
voltage
command
Patch-clamp: the special case of the
voltage clamp
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
Patch-clamp: the special case of the
voltage clamp
(2) “Gigaohm-seal”
R > 1 GOhm
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.
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
+
+
Cell
Patch-clamp: the special case of the
voltage clamp
closed
(3) Turn on the
aimed potential
the inside part of
the pipette and
keep it constantly
by applying the
voltage clamp
technique.
open
open
Cell
Properties of individual voltagedependent sodium channels
voltage command
10 msec
Properties of individual voltagedependent sodium channels
1. Individual channels are either open
or closed (no partial openings)
Properties of individual voltagedependent 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 voltagedependent 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
Properties of individual voltagedependent 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
Properties of individual voltagedependent 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
doesn’t open even once.
Summation of 300 recordings
The macroscopic
sodium current
Properties of individual voltagedependent 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
doesn’t open even once.
6. Second openings are rare (because
of inactivation)
Summation of 300 recordings
The macroscopic
sodium current
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
there’s no inactivation.
Slowly inactivating
K current channel
(Ram & Dagan,
1987)
The configurations of the patch-clamp technique
OnCell
Cell-Attached
The configurations of the patch-clamp
technique
OnCell
Insideout patch
The configurations of the patch-clamp technique
OnCell
Whole
Cell
The configurations of the patch-clamp technique
Whole
Cell
The configurations of the patch-clamp technique
Whole
Cell
outsideout patch
The whole-cell configuration
Rs
Rc
Cm
Extracellular solution (mM)
(for K currents)
NaCl 144
NaH2PO4 0.4
KCl 4
Intracellukar solution (mM)
(for K currents)
K-aspartate 100
KCl 25
K2HPO4 10,
K2EGTA 5
MgSO4 0.53
K2ATP 3
CaCl2 1.8
MgCl2 1
Glucose 5.5
HEPES 5
+
ICa blocker
HEPES 10
The whole cell configuration
Intracellular
solution
Patch-clamp amplifier
Micropipette
+
Extracellular
solution
_
_
_ +
+
_ _ +
+
+
_ _
+ +
_
+
_ _ +
Cell
10 ms ... 5000 ms
-20 mV ... +50 mV
-40 mV
IBM PC
The “run-down“ effect
The ATP-sensitive potassium current
The “run-down“
The L-type calcium current
The configurations of the patch clamp technique
Whole
Cell
Whole Cell, perforated
patch
- amphotericin-B
- nystatin
The “run-down”
The L-type calcium current
Cell isolation
- Ca2+ - free perfusion
- enzymatic digestion (collagenase)
- mechanical separation
L- type calcium current (ICa)
400 ms
-40 mV
200 pA
100 ms
0 mV
-40 mV
-35 mV
ICa amplítúdó (pA)
L- type calcium current (ICa)
0
-400
-800
-1200
-40
-20
0
20
Potenciál (mV)
40
60
-40 mV
-30 mV
ICa amplítúdó (pA)
L- type calcium current (ICa)
0
-400
-800
-1200
-40
-20
0
20
Potenciál (mV)
40
60
-40 mV
-25 mV
ICa amplítúdó (pA)
L- type calcium current (ICa)
0
-400
-800
-1200
-40
-20
0
20
Potenciál (mV)
40
60
-20 mV
-40 mV
ICa amplítúdó (pA)
L- type calcium current (ICa)
0
-400
-800
-1200
-40
-20
0
20
Potenciál (mV)
40
60
-15 mV
-40 mV
ICa amplítúdó (pA)
L- type calcium current (ICa)
0
-400
-800
-1200
-40
-20
0
20
Potenciál (mV)
40
60
-10 mV
-40 mV
ICa amplítúdó (pA)
L- type calcium current (ICa)
0
-400
-800
-1200
-40
-20
0
20
Potenciál (mV)
40
60
55 mV
-40 mV
ICa amplítúdó (pA)
L- type calcium current (ICa)
Current-voltage (I-V) relationship
0
-400
-800
-1200
-40
-20
0
20
40
Potenciál (mV)
Pre-incubation
5-10 min
drug
Wash-out
3-5 min
10-15 min
60