Title: CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES
1CELLULAR CARDIAC ELECTROPHYSIOLOGICAL TECHNIQUES
2Electrical model of the membrane Standard
intracellular microelectrode technique Voltage
clamp technique Patch clamp technique
3G1/R
4Ohms law
Ion channel model
5Current clamp
Voltage clamp
6Intracellular microelectrode technique
Re ltlt Rin Rin 1012 Ohm
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8Ag/AgCl 3 M KCl
Re 10 - 40 MOhm
0.1 - 0.2 ?m
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11The setup
amplifier
computer
ingerlo
A/D
d
e
r
Detected signal
P
0mV
Organ bath
d stimulating electrode
50 mV
e microelectrode
r referent electrode
100 ms
P preparation
120 mV
50
APA
Vmax
90
RP
APD50
APD90
drug
Wash-out
Pre-incubation
60 min
20-60 min
60 min
13Two microelectrode voltage clamp
test potential
voltage command
holding potential
The macroscopic sodium current
14The voltage-clamp circuit
voltage measure
follow up amplifier
voltage command
amplifier
Current measure
15Patch-clamp the special case of the voltage
clamp
16Patch-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
17Patch-clamp the special case of the voltage
clamp
(2) Gigaohm-seal
R gt 1 GOhm
Cell
18Patch-clamp the special case of the voltage
clamp
(3) Sense voltage here, inside the electrode, and
use voltage clamp to keep it constant.
Cell
19Patch-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
20Patch-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 voltage clamp technique.
closed open open
Cell
21Properties of individual voltage-dependent sodium
channels
voltage command
10 msec
22Properties of individual voltage-dependent sodium
channels
- Individual channels are either open or closed (no
partial openings)
23Properties of individual voltage-dependent sodium
channels
- Individual channels are either open or closed (no
partial openings) - 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
24Properties of individual voltage-dependent sodium
channels
- Individual channels are either open or closed (no
partial openings) - Each channel opening is only a brief event
compared to the total duration of the whole cell
voltage-dependent sodium current. - Channel opening and closing is variable in
duration and latency.
The macroscopic sodium current
25Properties of individual voltage-dependent sodium
channels
- The channels are either in open or closed state.
- The channel openings are short events when
compared with the macroscopic sodium current. - The time duration and latency of the channel
openings are variable (case sensitive). Might
happen to not open at all. - The open probability of the channels resembles
with that of the macroscopic current.
Summation of 300 recordings
The macroscopic sodium current
26Properties of individual voltage-dependent sodium
channels
- Individual channels are either open or closed (no
partial openings) - Each channel opening is only a brief event
compared to the total duration of the whole cell
voltage-dependent sodium current. - Channel opening and closing is variable in
duration and latency. - The overall probability of channel opening is
similar to the total sodium current. Look at the
sum of the currents from 300 trials. - Sometimes an individual channel doesnt open even
once.
Summation of 300 recordings
The macroscopic sodium current
27Properties of individual voltage-dependent sodium
channels
- Individual channels are either open or closed (no
partial openings) - Each channel opening is only a brief event
compared to the total duration of the whole cell
voltage-dependent sodium current. - Channel opening and closing is variable in
duration and latency. - The overall probability of channel opening is
similar to the total sodium current. Look at the
sum of the currents from 300 trials. - Sometimes an individual channel doesnt open even
once. - Second openings are rare (because of
inactivation)
Summation of 300 recordings
The macroscopic sodium current
28Similarly, individual potassium channels, calcium
channels, and other channels can be studied by
patch clamping
- Individual channels are either open or closed (no
partial openings). Sometimes more than one
channel is in a patch. - Each channel opening is only a brief event
compared to the total duration of the whole cell
current. - Channel opening and closing is variable in
duration and latency. - The overall probability of channel opening is
similar to the whole cell current - Second openings can happen if theres no
inactivation.
Slowly inactivating K current channel (Ram
Dagan, 1987)
29The configurations of the patch-clamp technique
On-Cell
Cell-Attached
30The configurations of the patch-clamp technique
Inside-out patch
On-Cell
31The configurations of the patch-clamp technique
Whole Cell
On-Cell
32The configurations of the patch-clamp technique
Whole Cell
33The configurations of the patch-clamp technique
outside-out patch
Whole Cell
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35The whole-cell configuration
Rs
Cm
Rc
36Intracellukar solution (mM) (for K currents)
Extracellular solution (mM) (for K currents)
NaCl 144 NaH2PO4 0.4 KCl 4 MgSO4 0.53 CaCl2
1.8 Glucose 5.5 HEPES 5 ICa blocker
K-aspartate 100 KCl 25 K2HPO4 10, K2EGTA 5 K2ATP
3 MgCl2 1 HEPES 10
37The whole cell configuration
Intracellular solution
Patch-clamp amplifier
Micropipette
IBM PC
_
_
_
Extracellular solution
_
_
_
Cell
_
_
_
_
10 ms ... 5000 ms
-20 mV ... 50 mV
-40 mV
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39The run-down effect
The ATP-sensitive potassium current
40The run-down
The L-type calcium current
41The configurations of the patch clamp technique
Whole Cell, perforated
patch
Whole Cell
- amphotericin-B - nystatin
42The run-down
The L-type calcium current
43Cell isolation
- Ca2 - free perfusion - enzymatic digestion
(collagenase) - mechanical separation
44L- type calcium current (ICa)
45L- type calcium current (ICa)
46L- type calcium current (ICa)
47L- type calcium current (ICa)
48L- type calcium current (ICa)
49L- type calcium current (ICa)
50L- type calcium current (ICa)
51L- type calcium current (ICa)
Current-voltage (I-V) relationship
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