心脏电生理技术基础(膜片钳)
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CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES
NORBERT JOST, PhD
2021/3/10
1
Electrical model of the membrane Standard intracellular microelectrode technique Voltage clamp technique Patch clamp technique
closed open
open
(3) Turn on the aimed potential the inside part of the pipette and keep it constantly by applying the voltage clamp technique.
2021/3/10
closed open
(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.
2021/3/10
++
Cell
19
Patch-clamp: the special case of the voltage clamp
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.
0mV
50 mV
d: stimulating electrode
e: microelectrode
r: referent electrode
100 ms
2P02:1/3/p10reparation
11
APA
Vmax
APD50
0 mV
50%
20 mV
100 ms
90% RP
APD90 Pre-incubation
6
2021/3/10
7
Ag/AgCl 3 M KCl
Re ~ 10 - 40 MOhm
0.1 - 0.2 m
2021/3/10Leabharlann 82021/3/10
9
2021/3/10
10
The setup
amplifier computer
ingerlő
d e
Pr
A/D Detected signal
Organ bath
The voltage-clamp circuit
follow up amplifier
voltage measure
2021/3/10
amplifier
voltage command
Current measure
14
Patch-clamp: the special case of the voltage clamp
60 min
2021/3/10
drug 20-60 min
Wash-out 60 min
12
Two microelectrode voltage clamp
test potential
voltage command
holding potential
2021/3/10
The macroscopic sodium cu1r3rent
1. Individual channels are either open or closed (no partial openings)
2021/3/10
22
Properties of individual voltagedependent sodium channels
1. Individual channels are either open or closed (no partial openings)
2021/3/10
2
G=1/R
2021/3/10
3
Ohm’s law
Ion channel model
2021/3/10
4
Current clamp Voltage clamp
2021/3/10
5
Intracellular microelectrode technique
2021/3/10
Re << Rin Rin = 1012 Ohm
2. Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current.
2021/3/10
The macroscopic sodium curren2t3
(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.
2021/3/10
Cell
18
Patch-clamp: the special case of the voltage clamp
2021/3/10
15
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)
2021/3/10
Cell
16
Patch-clamp: the special case of the voltage clamp
(2) “Gigaohm-seal”
R > 1 GOhm
2021/3/10
Cell
17
Patch-clamp: the special case of the voltage clamp
Cell
20
Properties of individual voltagedependent sodium channels
voltage command
10 msec
2021/3/10
21
Properties of individual voltagedependent sodium channels
NORBERT JOST, PhD
2021/3/10
1
Electrical model of the membrane Standard intracellular microelectrode technique Voltage clamp technique Patch clamp technique
closed open
open
(3) Turn on the aimed potential the inside part of the pipette and keep it constantly by applying the voltage clamp technique.
2021/3/10
closed open
(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.
2021/3/10
++
Cell
19
Patch-clamp: the special case of the voltage clamp
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.
0mV
50 mV
d: stimulating electrode
e: microelectrode
r: referent electrode
100 ms
2P02:1/3/p10reparation
11
APA
Vmax
APD50
0 mV
50%
20 mV
100 ms
90% RP
APD90 Pre-incubation
6
2021/3/10
7
Ag/AgCl 3 M KCl
Re ~ 10 - 40 MOhm
0.1 - 0.2 m
2021/3/10Leabharlann 82021/3/10
9
2021/3/10
10
The setup
amplifier computer
ingerlő
d e
Pr
A/D Detected signal
Organ bath
The voltage-clamp circuit
follow up amplifier
voltage measure
2021/3/10
amplifier
voltage command
Current measure
14
Patch-clamp: the special case of the voltage clamp
60 min
2021/3/10
drug 20-60 min
Wash-out 60 min
12
Two microelectrode voltage clamp
test potential
voltage command
holding potential
2021/3/10
The macroscopic sodium cu1r3rent
1. Individual channels are either open or closed (no partial openings)
2021/3/10
22
Properties of individual voltagedependent sodium channels
1. Individual channels are either open or closed (no partial openings)
2021/3/10
2
G=1/R
2021/3/10
3
Ohm’s law
Ion channel model
2021/3/10
4
Current clamp Voltage clamp
2021/3/10
5
Intracellular microelectrode technique
2021/3/10
Re << Rin Rin = 1012 Ohm
2. Each channel opening is only a brief event compared to the total duration of the whole cell voltage-dependent sodium current.
2021/3/10
The macroscopic sodium curren2t3
(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.
2021/3/10
Cell
18
Patch-clamp: the special case of the voltage clamp
2021/3/10
15
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)
2021/3/10
Cell
16
Patch-clamp: the special case of the voltage clamp
(2) “Gigaohm-seal”
R > 1 GOhm
2021/3/10
Cell
17
Patch-clamp: the special case of the voltage clamp
Cell
20
Properties of individual voltagedependent sodium channels
voltage command
10 msec
2021/3/10
21
Properties of individual voltagedependent sodium channels