CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES NORBERT JOST, PhD.
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Transcript of CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES NORBERT JOST, PhD.
Electrical model of the membrane
Standard intracellular microelectrode technique
Voltage clamp technique
Patch clamp technique
The setup
Organ bath
d: stimulating electrode
e: microelectrode
r: referent electrode
P: preparation
computer
A/D
ingerlő
amplifier
Detected signalP
de
r
100 ms
50 m
V
0mV
Two microelectrode voltage clamp
voltage command
holding potential
test potential
The macroscopic sodium current
The voltage-clamp circuit
voltage command
amplifier
Current measure
voltage measure
follow upamplifier
Cell
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
(3) Sense voltage here, inside the electrode, and use voltage clamp to keep it constant.
Patch-clamp: the special case of the voltage clamp
closed
open
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
open
Cell
(3) Turn on the aimed potential the inside part of the pipette and keep it constantly by applying the voltage clamp technique.
Patch-clamp: the special case of the voltage clamp
1. Individual channels are either open or closed (no partial openings)
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
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.
Properties of individual voltage-dependent sodium channels
The macroscopic sodium current
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.
Properties of individual voltage-dependent sodium channels
The macroscopic sodium current
Summation of 300 recordings
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
Properties of individual voltage-dependent sodium channels
The macroscopic sodium current
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
Properties of individual voltage-dependent sodium channels
The macroscopic sodium current
Slowly inactivating K current channel (Ram & Dagan, 1987)
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.
Similarly, individual potassium channels, calcium channels, and other channels
can be studied by patch clamping
NaCl 144NaH2PO4 0.4
KCl 4
MgSO4 0.53
CaCl2 1.8
Glucose 5.5
HEPES 5
+
ICa blocker
Intracellukar solution (mM)(for K currents)
Extracellular solution (mM)(for K currents)
K-aspartate 100
KCl 25
K2HPO4 10,
K2EGTA 5
K2ATP 3
MgCl2 1
HEPES 10
Extracellular solution
Patch-clamp amplifier
IBM PC
Micropipette
+ __
++
+ ++
_
_
__ _ ++
__
++_
Cell
-40 mV
-20 mV ... +50 mV10 ms ... 5000 ms
Intracellular solution
The whole cell configuration
Whole Cell
Whole Cell, perforated patch
- amphotericin-B- nystatin
The configurations of the patch clamp technique
-40 -20 0 20 40 60
-1200
-800
-400
0
I Ca
amp
lítú
dó
(p
A)
Potenciál (mV)
-35 mV-40 mV
L- type calcium current (ICa)
-40 -20 0 20 40 60
-1200
-800
-400
0
I Ca
amp
lítú
dó
(p
A)
Potenciál (mV)
-30 mV-40 mV
L- type calcium current (ICa)
-40 -20 0 20 40 60
-1200
-800
-400
0
I Ca
amp
lítú
dó
(p
A)
Potenciál (mV)
-25 mV-40 mV
L- type calcium current (ICa)
-40 -20 0 20 40 60
-1200
-800
-400
0
I Ca
amp
lítú
dó
(p
A)
Potenciál (mV)
-20 mV-40 mV
L- type calcium current (ICa)
-40 -20 0 20 40 60-1200
-800
-400
0
I Ca
amp
lítú
dó
(p
A)
Potenciál (mV)
-15 mV-40 mV
L- type calcium current (ICa)
-40 -20 0 20 40 60-1200
-800
-400
0
I Ca
amp
lítú
dó
(p
A)
Potenciál (mV)
-10 mV-40 mV
L- type calcium current (ICa)
-40 -20 0 20 40 60-1200
-800
-400
0
I Ca
amp
lítú
dó
(p
A)
Potenciál (mV)
55 mV
-40 mV
drug
5-10 min 3-5 min 10-15 min
Wash-outPre-incubation
Current-voltage (I-V) relationship
L- type calcium current (ICa)