The document provides an overview of the patch clamp technique. It discusses the history, principle, method, configurations, instrumentation, and applications of patch clamp. Some key points include: - Patch clamp allows the recording of currents through single ion channels in isolated patches of cell membrane. - It was developed in 1976 by Erwin Neher and Bert Sakmann, who received the Nobel Prize for this work. - Different configurations (whole-cell, outside-out, inside-out, cell-attached) are used depending on the question being studied. - It is used to identify ion channels and study their properties like activation, permeability, and modulation by drugs.

1. PATCH CLAMP
TECHNIQUE
Presented by:
Sandhya Talla
M.Pharm(Pharmacology)

2. Contents
Introduction
History
Principle
Method
Configurations
Instrumentation
Applications
References

3. INTRODUCTION
 Patch – A very minute portion of the cell membrane.
 Clamp – To hold a certain physical quantity to a constant value.
 It is an electrophysiological technique in which we are able to
CLAMP the VOLTAGE of an isolated piece of cell membrane (or
whole-cell) & observe CURRENTS that flow through ION
CHANNELS.
 It is the current that the patch-clamp amplifier supplies to hold the
What is Patch Clamp?
(Neher & Sakmann, 1991)

4.  Previously researcher only studies ion channels in
neurons collectively.
 By 1970 the terms Na+
channel & K+
channel were used
frequently, although there was no direct evidences for the
existence of ion channels available from biological
preparations.
(Muley, et al., 2009)

5. 1976 Erwin Neher and Bert Sakmann invented Patch Clamp.
1980 Serendipitous discovery of ‘Giga seal’.
1984 Noda et al. First time sequenced ion channels NAch and
Sodium channel.
1987 Tanabe et al. sequenced calcium channel.
1988 Kamb et al. sequenced Potassium channel.
1990 Fred Sigworth et al, and fertig et al. improved throughput by
introducing Planar Electrode Patch Clamp.
1991 Erwin Neher and Beert Sakmann earned The Nobel
Prize in Physiology or Medicine for patch clamp.

6.  1963 It was concluded Ohm’s law can be applied to biological
system,
(V=Change in potential ,I=Current ,R=Resistance of system)
Itotal = IC + Iionic [ where IC= Capacitive current,
Iionic = Ionic current. ]
When dV/dt=0,
Itotal = Iionic [As IC = C.(dV/dt)]
V=IR
PRINCIPLE
(Hille, 1970 & Neher, 1991)

7. Nernst Equation ,
Conc. Inside
EMF(Mili volt) = ± 61 log
Conc. Outside
Nernst & Goldman-Hodgkin-Katz (GHK) Equations
Goldman-Hodgkin-Katz (GHK) Equation,
CNA
+
I PNA
+
+ CK
+
I PK
+
CCl
-
I PCl
-
EMF(Mili volt) = - 61 log
CNA
+
O PNA
+
+ CK
+
O PK
+
CCl
-
O PCl
-

8. Patch Clamp Circuit
Patch of cell membrane with ion channel
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 GΩ
allowing very small currents (10-12
A)
to be measured.
(www.Googleimage.com)

9. Patch pipette

10. Patch clamp
configurations

15. open
closed
current
time
Closed stateClosed state
Open stateOpen state
Signal interpretation
(Neher, 1991)

16.  How clean the surface of the cell is that is under investigation
 If the membrane surface is ‘clean’ then recordings can be made
from almost any cell.
 The most popular preparations are:
Cultured cells
Acutely isolated cells
Cells in thin slices of brain tissue
Cells that have been transfected with cloned ion channels
Recent reports have documented patch clamp recordings
in vivo
Preparations used

17. 17
COMPUTER
PATCH CLAMP
TAPE
RECORDER
AIR TABLE
MOUNTING
HARDWARE
HEADSTAGE
OSCILLOSCOPE
MICROSCOPE
MICROMANIP-
ULATOR
FILTER
FARADAY CAGE
Block diagram
(Cahalan & Neher, 1992)

18. Significance of different configurations
(Petersen, 1986)

19.  Outside-out patch - Commonly used to study ligand- gated ion
channels as we can apply agonist to the external surface of the
membrane and hence gain access to the ligand binding site.
 Cell-attached patches - Used to study channels in their
‘normal’ environment.
Patch excision may result in modulation of channel properties.
Patch excision will also result in the loss of proteins/enzymes
Which configurations to be used?

20.  Inside-out patch - Commonly used to study the effects
of drugs that modulate the activity of a channel by interacting with
the intracellular domains of the channel.
 Whole-cell recording - Measures the activity in entire cell.

21. Current Conventions
Positive and negative currents
Inward and outward currents
In whole cell and outside out
Current clamp
In cell attached and inside out

22. Conventional patch clamp
Planar patch clamp
Perforated patch clamp
Loose patch clamping
Techniques
(David, 2001)

23. Identification of Ionic Channels:
Mode of activation
Neurotransmitter and Chemical analogues
Membrane Potential
Intracellular Ion Concentration
Secondary Messenger's
The Permeant Ions (Under Physiological Condition’s)
Unitary Conductance
Selective Block by Drugs, Ions or Toxins
Applications
(Neher, 1991)

24. Assay type Method Variants Advantages Disadvantages
Direct
functional
Extracellular
Voltage clamp
Multi-electrode
array
Patch-clamp
Two-electrode
voltage clamp
Measures cell
networks
Voltage
control,
highest
content,
gold standard
Same as above
No voltage
control, low
Throughput
Low–medium
throughput,
high cost
Same as above,
but lower
sensitivity
 Studying Potassium Channels:
Assay for potassium channel

25.  Ion-channel assay technologies
 Multimodal Distribution of Granule Sizes in Rat Mast Cells
 Combined patch-clamp technique and calcium measurement
 Channel Recording
 Expanded Scope of Electrophysiology

26.  Capacitance measurement
 Cell Signaling
 Detection of Macromolecular Translocation Through
Nuclear pores
 Automated Electrophysiology Assays

28. Avi P, et al. (2007) Ionic Requirements for Membrane-Glass
Adhesion and Giga Seal Formation in Patch-Clamp
Recording. Biophysical Journal 92, 3893–3900.
Bert S (1991) Elementary Steps in synaptic transmission
revealed by currents through single ion channels. Noble
lecture, 31-59.
David O and Peter S. Patch clamp techniques for single
channel and whole-cell recording Chapter 4, 53-78.
REFERENCES

29. Frederick S and Feng Q (1993) Gated, Ion-selective Channels
Observed with Patch Pipettes in the Absence of Membranes:
Novel Properties of a Gigaseal. Biophysical Journal 65, 1101-
1107.
Hodgkin A.L. and Huxley A.F (1952) Currents carried by
sodium and potassium ions through the membrane of the giant
axons of loligo. J.Physiol 116, 449-472.
Huxley A.F (1963) The quantitative analysis of excitation and
conduction in nerve. NobelLecture, 52-69.
Jia Xu, et al. (2004) High-throughput technologies for studying
potassium channels progresses and challenges, 32-38.

30. Kim D (2008) Screening assays for ion channels, Ion Channels
in drug discovery-focus on biological assays, 24-27.
Muley N, et al. (2009) Historical Events in Electrophysiology.
Currents Research & Information on Pharmaceuticals Sciences
10(1).
Neher E (1991) Ionic channels for communication between and
within cells. Nobel Lecture,10-25.
Sakmann B (1992) Elementary steps in synaptic transmission
revealed by currents through single ion channels. Neuron 8,
613-629.

31. Sakmann B, et al. (1984) Patch clamp techniques for studying
ionic channels in excitable membranes. Annual Review of
Physiology 46, 455-472.
Wyllie DJA (2007) Single-channel recording. In Patch-Clamp
Analysis – Advanced Techniques 2nd
Ed, 69-129.
WEB SITES
www.nobelprize.org
www.bio.psu.edu
www.scholargoogle.com
www.sciencedirect.com
www.pubmed.com