patch clamp technique
TRANSCRIPT
PATCH CLAMP TECHNIQUE
BY- RITIK VARDHANM.Sc.(P)
ROLL NO.- 1366
INTRODUCTION HISTORY PRINCIPLE CONFIGURATION & TYPES APPLICATION LIMITATION RECENT RESEARCH
CONTENTS
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 .
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INTRODUCTION
Jan Swammerda
m
• earliest experiments in electrophysiology
Luigi Galvani
• the first experimental evidence of electrical activity in animals by using metal wires in frog muscle
Hodgkin and Huxley
• the first intracellular measurement of the action potential in the giant squid axon
• Impaling micropipettes developed by skeletal muscle fibres
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HISTORICAL DEVELOPMENT
Graham
Cole and Marmont
• Voltage clamp technique combined with micropipettes
Sakmann and
Neher
• the patch clamp technique
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Continues………………….
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.
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NEED OF PATCH CLAMP
THE PATCH-CLAMP TECHNIQUE
Erwin NeherBert Sakmann
Germany(1991 Nobel Laureates)
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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
fire -polished glass pipette
Electrolyte solution
Electrode (10-25 µm)
<10nm
10 GΩ resistor at 20°C, the standarddeviation of the current noise at 1 kHz will be 0.04 pA,8
The patch-clamp circuit
Patch of cell membrane with ion channel
FBR
_
+Amplifier
TechnicalThe high gain operational amplifier isconnected in the circuit so that the currentflowing through the ion channel is measuredas a voltage drop across the feedback resistor(FBR). The FBR has a resistance of 50 Gallowing very small currents (10-12 A)to be measured.
A patch-clamp rig
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CONFIGURATION OF PATCH CLAMP:
• On-cell
• Inside Out
• Whole Cell
• OutsideOut
Perforated patch
Loose patch
DPerforated-patch method (simplified)ASF
TYPES
Patch clamp technique in isolated cardiac myocytes
Perfusion of a section of intact canine left ventricularmyocardium. A cannula has been placed into theleft anterior descending coronary artery and clampshave been placed to occlude major coronary arterybranches that have been transected during sectioning
Male wistar rat
ISOLATION OF MYOCYTES
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.
PRINCIPLE & PROCEDURE
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
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.
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.
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.
EVALUATION
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
limitations
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.
APPLICATIONS
Measurements are conducted in amultiparametric
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
RECENT RESEARCH
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.
conclusion
References1. Wyllie DJA (2007) Single-channel recording. In Patch-Clamp Analysis –
Advanced Techniques 2nd Edition. pp 69-129. Ed. Walz W. Humana Press Totowa, New Jersey USA
2. 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.