1. BY- RITIK VARDHAN
M.Sc.(P)
ROLL NO.- 1366

2. CONTENTS

 INTRODUCTION
 HISTORY
 PRINCIPLE
 CONFIGURATION & TYPES
 APPLICATION
 LIMITATION
 RECENT RESEARCH

3. INTRODUCTION

 The patch clamp technique is a laboratory technique
in electrophysiology that allows the study of single or
multiple ion channels in cells.
 Sakmann and Neher - develop the patch clamp technique
in 1970s and early 1980s.
 Received the Nobel prize for this high scientificwork
in1991 .
3

4. HISTORICAL DEVELOPMENT
Jan
Swammerda
m
Luigi
Galvani 
Hodgkin and
Huxley Graham
• earliest • the first • the first • Impaling
experiment experiment intracellula micropipett
s in al evidence r es
electrophys of electrical measureme developed
iology activity in nt of the by skeletal
animals by action muscle
using metal potential in fibres
wires in the giant
frog muscle squid axon
4

5. Continues………………….
Cole and Marmont  Sakmann and
Neher
• Voltage clamp • the patch clamp
technique technique
combined with
micropipettes
5

6. NEED OF PATCH CLAMP

 Patch clamp is refinement of voltage clamp technique.
 provides for low-noise recordings of current
 Provides access to the inside of the cell
 Can insert an electrode into the cell
 Can change the intracellular fluid
 Creates a seal impermeable to ion flow
 High electrical resistance
 Allows one to measure current through ion channels vs.
voltage, time, temperature.
6

7. THE PATCH-CLAMP TECHNIQUE
 Erwin Neher
Bert Sakmann
Germany
(1991 Nobel Laureates)
7

8. 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 with a suitable electrolyte solution, against the surface of a cell and
applying light suction
Electrode (10-25 µm)
fire -polished glass pipette
Electrolyte solution
10 GΩ resistor at 20°C, the standard
7/7/2011 8
deviation of the current noise at 1 kHz will be 0.04 pA,
8
<10nm

9. The patch-clamp circuit
FBR
_
+
Amplifier
Technical
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 50 G
allowing very small currents (10-12 A)
to be measured.
Patch of cell membrane with ion channel

10. A patch-clamp rig


11. CONFIGURATION OF PATCH CLAMP:
• On-cell 
• Inside Out
• Whole Cell
• OutsideOut
11

12. 

13. TYPES

 Perforated patch
 Loose patch
DPerforated-patch method
(simplified)ASF

14. Patch clamp technique in isolated cardiac
myocytes

Perfusion of a section of intact canine 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

15. ISOLATION OF MYOCYTES
 Male wistar rat


16. PRINCIPLE &
PROCEDURE

 The generation of an action potential in heart 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 experiment.
 Glass micro-pipette - a tip opening of about 1 μm, is
placed onto the cell.

17.  The patch-pipette is filled with either high 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 electronical amplifier.
 A second chlorided silver wire is inserted into the bath and
serves a ground electrode.
 Whole cell patch clamping is done

18. This high input resistance enables the recording of small electrical
currents in the range of Picosiemens (10–12 S), which are flowing

through channel-forming proteins situated in the membrane patch.
The electrical current is driven by applying an electrical potential
across the membrane patch, and/or by establishing an appropriated
chemical gradient for the respective ion species.

19. 
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).
 The different types of K+ channels existing in cardiomyocyte.

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

21. limitations

 Imparting skillful training performance and recording
In during single channel recordings
 Cost of process is expensive
 Time consuming
 Number of samples required is more at times
 Chance of membrane distortion

22. APPLICATIONS
 For the evaluation of antiarrhythmics agents.
 In kidney cells.
 
Used for isolated ventricular myocytes from Guinea pigs to study a cardio
selective inhibition of the ATP sensitive potassium channel.
 To identify multiple types of calcium channels.
 To measure the effect of potassium channel openers.
 Used in the molecular biology.
 Voltage clamp studies on sodium channels.
 Used to investigate a wide range of electrophysiological cell properties.
 Measurement of cell membrane conductance.

23. RECENT RESEARCH
amultiparametric
 Measurements are conducted in
manner in an integrated and automated microfluidic
chip.
 Micropippetes in traditional patch clamp technique are
replaced by nano machine patch clamp system with
integrated micro fluidics which aids
Rapid Intra cellular perfusion
Improved optical measurments
Rapid measurment of single cell dose response curves

24. conclusion

 It is higly modified and successful technique
 Development of this technique is being done for newer
approaches to yield better accurate and efficient
information which aids drug discovery process.

25. References
1. Wyllie DJA (2007) Single-channel recording. In Patch-Clamp Analysis –
Advanced Techniques 2nd Edition. pp 69-129. Ed. Walz W. Humana Press
2.
Totowa, New Jersey USA

Sakmann B (1992) Elementary steps in synaptic transmission revealed by
currents through single ion channels. Neuron 8, 613-629.
This is his 1992 Nobel Lecture and well worth a read
3. Aidley DJ & Stanfield PR (1996) Ion Channels – Molecules in Action
Cambridge University Press.
This is a very good introductory textbook.