The patch-clamp technique allows the study of single ion channels in cells. It was developed in the late 1970s and early 1980s by Erwin Neher and Bert Sakmann, who received the Nobel Prize for this work. There are different variations of the patch-clamp technique that provide access to the inside or outside of the cell membrane to study channel properties under various conditions. The technique uses a glass pipette pressed against a cell to form a high resistance seal and then precisely measures electric currents flowing through individual or multiple ion channels.

1. Patch-clamp
technique
HOVSEP GHAZARYAN

2. Introduction
 The patch clamp technique is a laboratory
technique in electrophysiology that allows the
study of single or multiple ion channels in cells.
 The patch clamp technique is a refinement of
the voltage clamp. Erwin Neher and Bert
Sakmann developed the patch clamp in the
late 1970s and early 1980s.
 Neher and Sakmann received the Nobel Prize in
Physiology or Medicine in 1991 for this work.

3. Applications
Neurons
Cardiomyocytes
Muscle fibers
Panceratic betta cells
Bacteria
Kidney cells

4. Applications
For evaluation of antiarrythmics
agents
To study a cardio selective
inhibition of ATP sensitive
potassium channel
To identify multiple types of
calcium channels

5. Applications
To measure the effect of potassium
channel openers
Voltage clamp studies on sodium
channels
To investigate wide range of
electropysiological cell properties
Measurement of cell membrane
conductance

6. Historical development
Swammerdam
• Earliest
experiments in
electrophysiology
Galvani
• The first
experimental
evidence of
electrical activity
in animals by
using metal wires
in frog muscle
Hodgkin & Huxley
• The first
intracellular
measurement of
the action
potential in the
giant squid axon

7. Historical development
Graham
•Impaling
micropipettes
developed
by skeletal
muscle fibers
Cole &
Marmont
•Voltage
clamp
combined
with
micropipettes
Sakmann &
Neher
•The patch-
clamp
technique

8. Need of patch-clamp
Patch-clamp is refinement of voltage clamp technique
Provides for low-noise recordings of currents
Provides access to the inside of cell
•Can insert an electrode into the cell
•Can change intracellular fluid
Creates a seal impermeable to ion flow
•High electrical resistance
Allows one to measure current through ion channels vs. voltage, time, temperature

9. The patch-clamp technique

10. Basic principle

11. Basic principle
 The principle of the method is to isolate a patch of
membrane electrically from the external solution and to
record current flowing into the patch
 This is achieved by pressing a fire-polished glass pipette,
which has been filled wit suitable electrolyte solution,
against the surface of a cell and applying light suction
 10GΩ resistor at 20C, the standard deviation of the
current noise at 1kHz will be 0.04pA

12. The patch-clamp circuit

13. The patch-clamp circuit
 The high gain operational amplifier is
connected in the circuit so that the
current flowing through the ion channel is
measured as a voltage drop across the
feedback resistor (FBR). The FBR has a
resistance of 50GΩ allowing very small
currents (10-12A) to be measured

14. Cell
Patch-clamp
Suck a small piece of
membrane onto the
tip of a glass
micropipette
(~ 1 µm in diameter)

15. Cell
“Gigaohm-seal”
R > 1 GOhm
Patch-clamp

16. Cell
Sense voltage
here, inside the
electrode, and
use voltage clamp
to keep it
constant.
Patch-clamp

17. closed
open
Cell
+ +
Patch-clamp
Sense voltage
here, inside the
electrode, and
use voltage clamp
to keep it
constant.

18. closed closed
open
Cell
Turn on the aimed
potential the inside
part of the pipette
and keep it
constantly by
applying the voltage
clamp technique.
Patch-clamp

19. Recording

20. Recording
 Many patch clamp amplifiers do not use
true voltage clamp circuitry, but instead are
differential amplifiers that use the bath
electrode to set the zero current (ground) level.
Current is then injected into the system to
maintain a constant, set voltage.
 Alternatively, the cell can be current clamped in
whole-cell mode, keeping current constant
while observing changes in membrane voltage.

21. voltage command
10 msec
Properties of individual voltage-
dependent sodium channels

22. 1. Individual channels are either open or closed
(no partial openings)
Properties of individual voltage-
dependent sodium channels

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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.
Other channels

29. Variations of patch-clamp
 Cell-attached patch
 Inside-out patch
 Whole-cell recording or whole-cell patch
 Outside-out patch
 Perforated patch
 Loose patch

30. Cell-attached patch

31. Cell-attached patch
 Allows the recording of currents through single, or a few, ion
channels contained in the patch of membrane captured by the
pipette. By not disrupting the interior of the cell, any intracellular
mechanisms normally influencing the channel will still be able to
function as they would physiologically.
 The technique is thus limited to one point in a dose response
curve per patch.
 Voltage-gated ion channels can be clamped successively at
different membrane potentials in a single patch.

32. Inside-out patch

33. Inside-out patch
The experimenter has access to
the intracellular surface of the
membrane via the bath and can
change the chemical
composition of what the surface
of the membrane is exposed to.

34. Whole-cell patch

35. Whole-cell patch
 Larger opening at the tip of the patch clamp
electrode provides lower resistance and thus
better electrical access to the inside of the cell.
 Because the volume of the electrode is larger
than the volume of the cell, the soluble contents
of the cell's interior will slowly be replaced by the
contents of the electrode.

36. Outside-out patch

37. Outside-out patch
 Complementarity to the inside-out technique.
 Places the external rather than intracellular
surface of the cell membrane on the outside of
the patch of membrane, in relation to the patch
electrode.
 The longer formation process involves more steps
that could fail and results in a lower frequency of
usable patches.

38. Perforated patch

39. Perforated patch
 Similar to the whole-cell configuration
 Suction is not used to rupture the patch membrane
 The electrode solution contains small amounts of an
antifungal or antibiotic agent, which diffuses into the
membrane patch and forms small pores in the
membrane
 The perforated patch can be likened to a screen door
that only allows the exchange of certain molecules from
the pipette solution to the cytoplasm of the cell

40. Loose patch

41. Loose patch
 Employs a loose seal (low electrical resistance) rather
than the tight gigaseal used in the conventional
technique.
 The pipette is moved slowly towards the cell, until the
electrical resistance of the contact between the cell.
and the pipette increases to a few times greater
resistance than that of the electrode alone.
 The pipette that is used can be repeatedly removed
from the membrane after recording, and the membrane
will remain intact.

42. Rs
CmRc
Whole-cell configuration

43. NaCl 144
NaH2PO4 0.4
KCl 4
MgSO4 0.53
CaCl2 1.8
Glucose 5.5
HEPES 5
+
ICa blocker
Intracellular 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

44. Extracellular
solution
Patch-clamp amplifier
IBM PC
Micropipette
+ _
_
+
+
+ +
+
_
_
_
_ _
++
_
_
+
+_
Cell
-40 mV
-20 mV ... +50 mV
10 ms ... 5000 ms
Intracellular
solution
Whole cell configuration

45. Patch-clamp technique in isolated
cardiac myocytes
 Perfusion of section of intact
left ventricular myocardium. A
cannula has been placed into
the left anterior descending
coronary artery and clamps
have been placed to occlude
major coronary artery
branches that have been
transected during sectioning

46. Isolation of myocytes
 37C
 Male wistar rat
Intact heart Section
Wedge
preparation
Perfusion (Physiologic saline → Ca2+
free saline → Ca2+ free enzyme)
Dissection
Epicardial preparation Batch digestion Filtration
Isolated myocytes Incubation buffer (0.5M Ca2+
→ 1M Ca2+ )
Electraphysiologic study

47. Principle & procedure
 The generation of an action potential in hearth
muscle cells depends on the opening and
closing of ion-selective channels in the plasma
membrane
 The patch-clamp technique enables the
investigation of drug interactions with ion-
channel
 The isolated cells are ready for experiments
 Glass micro-pipette - a tip opening of about 1
µm, is placed onto the cell

48. Principle & procedure
 The patch-pipette is filled with either NaCL or KCl
solution and is mounted on a micro manipulator
 A chlorided silver wire connects the pipette
solution to the head stage of an electrical
amplifier
 A second chlorided silver wire is inserted into the
bath and serves a ground electrode
 Whole cell patch clamping is done

49. Principle & procedure
 High input resistance enables the recording of
small electrical currents, which are flowing
through channel forming proteins in the
membrane patch
 The electrical current is driven by applying an
electrical potential across membrane patch,
and/or by establishing an appropriated
chemical gradient for the respective ion species

50. Principle & procedure
 It is important to investigate the interaction of
drugs with all ion channels involved in the
functioning of the heart muscle cell (K+, Na+,
Ca2+ and eventually Cl- channels)

51. Evaluation
 Concentration-response curves of drugs which
either inhibit or activate ion channels can be
recorded either on he single channel level or by
measuring the whole-cell current
 IC50 and EC50 values (50% inhibition or
activation, respectively) can be obtained

52. Limitations
 Requires strong background in ion channel
biophysics
 Imparting skillful training performance during
single channel recordings
 Cost of process is expensive
 Time consuming
 Number of samples required is more at times
 Chance of membrane distortion

53. Conclusion
Patch-clamp is highly modified and
successful technique
Development of this technique is
being done for newer approaches
to yield accurate and efficient
information which aids drug
discovery process.

54. Thank you