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
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
Cell
Patch-clamp: the special case of the voltage clamp
(3) Sense
closed
open
voltage here, inside the electrode, and use voltage clamp to keep it constant.
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.
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.
Summation of 300 recordings
The macroscopic sodium current
Similarly, individual potassium channels, calcium channels, and other channels can be studied by patch clamping
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)
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
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.
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.
CELLULAR CARDIAC
ELECTROPHYSIOLOGICAL TECHNIQUES
NORBERT JOST, PhD
Electrical model of the membrane
Standard intracellular microelectrode technique Voltage clamp technique Patch clamp technique
closed
open
open
Cell
Properties of individual voltagedependent sodium channels
voltage command
10 msec
Properties of individual voltagedependent sodium channels
0.1 - 0.2 m
The setup
amplifier
ingerlő d P A/D e r Detected signal computer
Organ bath d: stimulating electrode e: microelectrode
0mV
r: referent electrode
+
Cell
+
Patch-clamp: the special case of the voltage clamp
(3) Turn on the aimed potential the inside part of the pipette and keep it constantly by applying the voltHale Waihona Puke ge clamp technique.
P: preparation
50 mV 100 ms
0 mV
APA Vmax
50%
20 mV 100 ms
90%
RP APD50 APD90 Pre-incubation drug Wash-out
60 min
20-60 min
60 min
Two microelectrode voltage clamp
test potential
voltage command