Download CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES

CELLULAR CARDIAC
ELECTROPHYSIOLOGICAL TECHNIQUES
NORBERT JOST, PhD
Electrical model of the membrane
Standard intracellular microelectrode technique
Voltage clamp technique
Patch clamp technique
1
G=1/R
Ohm’s law
Ion channel model
2
Current clamp
Voltage clamp
Intracellular microelectrode technique
Re << Rin
Rin = 1012 Ohm
3
Ag/AgCl
3 M KCl
Re ~ 10 - 40 MOhm
0.1 - 0.2 µm
4
5
The setup
amplifier
computer
ingerlı
d
A/D
e
P
r
Detected signal
0mV
Organ bath
50 mV
d: stimulating electrode
e: microelectrode
r: referent electrode
100 ms
P: preparation
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
6
Two microelectrode voltage clamp
test potential
voltage command
holding potential
The macroscopic sodium current
The voltage-clamp circuit
follow up
amplifier
amplifier
voltage
measure
voltage
command
Current
measure
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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
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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
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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
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
10
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)
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
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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
there’s no inactivation.
Slowly inactivating
K current channel
(Ram & Dagan,
1987)
The configurations of the patch-clamp
technique
OnCell
Insideout patch
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The configurations of the patch-clamp technique
OnCell
Whole
Cell
The configurations of the patch-clamp technique
Whole
Cell
outsideout patch
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The whole-cell configuration
Rs
Rc
Cm
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Extracellular solution (mM)
(for K currents)
Intracellukar solution (mM)
(for K currents)
NaCl 144
NaH2PO4 0.4
K-aspartate 100
KCl 4
K2HPO4 10,
KCl 25
K2EGTA 5
MgSO4 0.53
K2ATP 3
CaCl2 1.8
MgCl2 1
Glucose 5.5
HEPES 10
HEPES 5
+
ICa blocker
The whole cell configuration
Intracellular
solution
Micropipette
+
Extracellular
solution
Patch-clamp amplifier
_
_
_ +
+
IBM PC
_ _ +
+
+
_ _
+ +
_
+ +
_ _
Cell
10 ms ... 5000 ms
-20 mV ... +50 mV
-40 mV
15
The “run-down“ effect
The ATP-sensitive potassium current
16
The “run-down“
The L-type calcium current
The configurations of the patch clamp technique
Whole
Cell
Whole Cell, perforated
patch
- amphotericin-B
- nystatin
17
The “run-down”
The L-type calcium current
Cell isolation
- Ca2+ - free perfusion
- enzymatic digestion (collagenase)
- mechanical separation
18
L- type calcium current (ICa)
400 ms
0 mV
-40 mV
200 pA
100 ms
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
60
Potenciál (mV)
Pre-incubation
5-10 min
drug
Wash-out
3-5 min
10-15 min
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