心脏电生理技术基础(膜片钳)
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.
G=1/R
.
Ohm’s law
Ion channel model
.
Current clamp Voltage clamp
.
Intracellular microelectrode technique
Re << Rin Rin = 1012 Ohm
.
.
Baidu Nhomakorabea
Ag/AgCl 3 M KCl Re ~ 10 - 40 MOhm
C. ell
Patch-clamp: the special case of the voltage clamp
(2) “Gigaohm-seal”
R > 1 GOhm
C. ell
Patch-clamp: the special case of the voltage clamp
(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.
0.1 - 0.2 m .
.
.
The setup
amplifier computer
ingerlő
d e
Pr
A/D Detected signal
Organ bath
d: stimulating electrode e: microelectrode r: referent electrode P: preparation
The macroscopic
.
sodium current
Properties of individual voltagedependent sodium channels
C. ell
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)
50 mV
0mV
100 ms .
APA
Vmax
APD50
0 mV
50%
20 mV
100 ms
90% RP
APD90 Pre-incubation
60 min
drug 20-60 min
.
Wash-out 60 min
Two microelectrode voltage clamp
test potential
++
C. ell
Patch-clamp: the special case of the voltage clamp
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.
C. ell
Patch-clamp: the special case of the voltage clamp
closed open
(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.
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.
voltage command
holding potential
. The macroscopic sodium current
The voltage-clamp circuit
follow up amplifier
voltage measure
amplifier
voltage command
Current measure
.
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.
.
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)
CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES
NORBERT JOST, PhD
.
Electrical model of the membrane Standard intracellular microelectrode technique Voltage clamp technique Patch clamp technique
The macroscopic
.
sodium current
Properties of individual voltagedependent sodium channels
1. Individual channels are either open or closed (no partial openings)
G=1/R
.
Ohm’s law
Ion channel model
.
Current clamp Voltage clamp
.
Intracellular microelectrode technique
Re << Rin Rin = 1012 Ohm
.
.
Baidu Nhomakorabea
Ag/AgCl 3 M KCl Re ~ 10 - 40 MOhm
C. ell
Patch-clamp: the special case of the voltage clamp
(2) “Gigaohm-seal”
R > 1 GOhm
C. ell
Patch-clamp: the special case of the voltage clamp
(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.
0.1 - 0.2 m .
.
.
The setup
amplifier computer
ingerlő
d e
Pr
A/D Detected signal
Organ bath
d: stimulating electrode e: microelectrode r: referent electrode P: preparation
The macroscopic
.
sodium current
Properties of individual voltagedependent sodium channels
C. ell
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)
50 mV
0mV
100 ms .
APA
Vmax
APD50
0 mV
50%
20 mV
100 ms
90% RP
APD90 Pre-incubation
60 min
drug 20-60 min
.
Wash-out 60 min
Two microelectrode voltage clamp
test potential
++
C. ell
Patch-clamp: the special case of the voltage clamp
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.
C. ell
Patch-clamp: the special case of the voltage clamp
closed open
(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.
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.
voltage command
holding potential
. The macroscopic sodium current
The voltage-clamp circuit
follow up amplifier
voltage measure
amplifier
voltage command
Current measure
.
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.
.
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)
CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES
NORBERT JOST, PhD
.
Electrical model of the membrane Standard intracellular microelectrode technique Voltage clamp technique Patch clamp technique
The macroscopic
.
sodium current
Properties of individual voltagedependent sodium channels
1. Individual channels are either open or closed (no partial openings)