1. The patch clamp technique allows the study of single or multiple ion channels in cells by isolating a patch of cell membrane using a glass pipette. 2. Erwin Neher and Bert Sakmann developed the patch clamp technique in the late 1970s and received the Nobel Prize for this work in 1991. 3. The patch clamp technique can be used to study ion channels in neurons, muscle cells, pancreatic cells, bacteria, and other cell types. It provides insights into drug effects on ion channels and cellular electrophysiology.

1. D R . R E S HMA K O T I A N,
M S C - CR
Patch-clamp technique

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

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

21. 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

22. 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

23. 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

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.
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

25. 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

26. 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

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

28. Cell-attached patch

29. 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.

30. Inside-out patch

31. 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.

32. Whole-cell patch

33. 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.

34. Outside-out patch

35. 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.

36. Perforated patch

37. 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

38. Loose patch

39. 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.

40. Rs
CmRc
Whole-cell configuration

41. 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

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

43. 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

44. 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

45. 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

46. 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

47. 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

48. 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)

49. 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

50. 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

51. 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.

52. Thank you