Patch Clamp Techniques

CELLULAR CARDIAC

ELECTROPHYSIOLOGICAL TECHNIQUES
NORBERT JOST, PhD
Electrical model of the membrane
Standard intracellular microelectrode technique
Voltage clamp technique
Patch clamp technique
G=1/R
Ohms law Ion channel model
Current clamp
Voltage clamp
Intracellular microelectrode technique
R
e
<< R
in

R
in
= 10
12
Ohm

0.1 - 0.2 m
Ag/AgCl
3 M KCl
R
e
~ 10 - 40 MOhm
The setup
Organ bath
d: stimulating electrode
e: microelectrode
r: referent electrode
P: preparation
computer
A/D
ingerl
amplifier
Detected signal
P
d
e
r
100 ms
5
0

m
V

0mV
drug
60 min 20-60 min 60 min
0 mV
20 mV
100 ms
APA
RP
APD
50

APD
90

90%
50%
V
max

Wash-out Pre-incubation
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 up
amplifier

Patch-clamp: the special case of the
voltage clamp

Cell
voltage clamp
(1) Suck a small
piece of
membrane onto
the tip of a glass
micropipette
(~ 1 m in
diameter)

Cell
(2) Gigaohm-seal
R > 1 GOhm
voltage clamp

Cell
(3) Sense
voltage here,
inside the
electrode, and
use voltage
clamp to keep
it constant.
voltage clamp
closed

open

Cell
+ +
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.
voltage clamp
voltage command
10 msec
Properties of individual voltage-
dependent sodium channels
1. Individual channels are either open
or closed (no partial openings)
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
1. Individual channels are either open or
closed (no partial openings)
sodium current.
3. Channel opening and closing is
variable in duration and latency.
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.
The macroscopic
sodium current
Summation of 300 recordings
sodium current.
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
doesnt open even once.

The macroscopic
sodium current
sodium current.
opening is similar to the total sodium
current. Look at the sum of the
currents from 300 trials.
5. Sometimes an individual channel
doesnt open even once.
6. Second openings are rare (because
of inactivation)

The macroscopic
sodium current
Slowly inactivating
K current channel
(Ram & Dagan,
1987)
closed (no partial openings).
Sometimes more than one channel is
in a patch.
of the whole cell current.
opening is similar to the whole cell
current
5. Second openings can happen if
theres no inactivation.
Similarly, individual potassium channels,
calcium channels, and other channels
can be studied by patch clamping

On-
Cell
Cell-Attached
The configurations of the patch-clamp technique

On-
Cell
Inside-
out patch
The configurations of the patch-clamp
technique

Whole
Cell
On-
Cell

Whole
Cell

Whole
Cell
outside-
out patch
R
s

C
m

R
c

The whole-cell configuration
NaCl 144
NaH
2
PO
4
0.4

KCl 4

MgSO
4
0.53

CaCl
2
1.8

Glucose 5.5

HEPES 5

+
I
Ca
blocker
Intracellukar solution (mM)
(for K currents)
Extracellular solution (mM)
(for K currents)
K-aspartate 100
KCl 25
K
2
HPO
4
10,
K
2
EGTA 5
K
2
ATP 3

MgCl
2
1

HEPES 10

Extracellular
solution
Patch-clamp amplifier
IBM PC
Micropipette
+ _
_
+
+
+ +
+
_
_
_
_
_
+
+
_
_
+
+
_
Cell
-40 mV
-20 mV ... +50 mV
10 ms ... 5000 ms
Intracellular
solution
The whole cell configuration
The run-down effect
The ATP-sensitive potassium current
The L-type calcium current
The run-down

Whole
Cell
Whole Cell, perforated
patch
- amphotericin-B
- nystatin
The configurations of the patch clamp technique
The run-down
The L-type calcium current
Cell isolation
- Ca
2+
- free perfusion

- enzymatic digestion (collagenase)

- mechanical separation
200 pA
100 ms
0 mV
400 ms
-40 mV
L- type calcium current (I
Ca
)
-40 -20 0 20 40 60
-1200
-800
-400
0
I
C
a

a
m
p
l

(
p
A
)
Potencil (mV)
-35 mV
-40 mV
Ca
)
-40 -20 0 20 40 60
-1200
-800
-400
0
I
C
a

a
m
p
l

(
p
A
)
Potencil (mV)
-30 mV
-40 mV
Ca
)
-40 -20 0 20 40 60
-1200
-800
-400
0
I
C
a

a
m
p
l

(
p
A
)
Potencil (mV)
-25 mV
-40 mV
Ca
)
-40 -20 0 20 40 60
-1200
-800
-400
0
I
C
a

a
m
p
l

(
p
A
)
Potencil (mV)
-20 mV
-40 mV
Ca
)
-40 -20 0 20 40 60
-1200
-800
-400
0
I
C
a

a
m
p
l

(
p
A
)
Potencil (mV)
-15 mV
-40 mV
Ca
)
-40 -20 0 20 40 60
-1200
-800
-400
0
I
C
a

a
m
p
l

(
p
A
)
Potencil (mV)
-10 mV
-40 mV
Ca
)
-40 -20 0 20 40 60
-1200
-800
-400
0
I
C
a

a
m
p
l

(
p
A
)
Potencil (mV)
55 mV
-40 mV
drug
5-10 min 3-5 min 10-15 min
Wash-out Pre-incubation
Current-voltage (I-V) relationship
Ca
)

Patch Clamp Techniques

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CELLULAR CARDIAC

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