patch clamp ppt by kp
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my seminar topicTRANSCRIPT
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PATCH CLAMP TECHNIQUE
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UNDER THE GUIDENCE OF
MISS MEENU SINGH
ASST.PROFEESOR
DEPARTMENT OFPHARMACOLOGY
CMRCP
BY
KRISHNAPRIYA.P
M.PHARM
PHARMACOLOGY
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 .
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HISTORICAL DEVELOPMENT
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Graham
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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 cellCan change the intracellular fluid
Creates a seal impermeable to ion flowHigh electrical resistance
Allows one to measure current through ion channels vs. voltage, time, temperature.
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THE PATCH-CLAMP TECHNIQUE
Erwin NeherBert Sakmann
Germany(1991 Nobel Laureates)
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BASIC PRINCIPLE7
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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)
Renitence
Si 2 = 4kTfc /R
variance of the current noise (in A2) k = Boltzmann’s constant, T = temperature (° Kelvin), fc = the bandwidth(Hz)
<10nm
10 GΩ resistor at 20°C, the standarddeviation of the current noise at 1 kHz will be 0.04 pA,8
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TYPES OF PATCH CLAMP:
• On-cell
• Inside Out
• Whole Cell
• OutsideOut
TYPES
Perforated patch
Loose patch
Blind patch
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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
ISOLATION OF MYOCYTES
Male wistar rat
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.
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
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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
wholecell
current. IC50 and EC50 values (50% inhibition or
activation, respectively) can be obtained
LIMITATIONS
Requires strong background inc channel biophysics
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
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.
RECENT RESEARCH
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
Large experimental through put
CONCLUSION
It is higly modified and successful technique
Developmant of this technique is being done for newer
approaches to yield better accurate and efficient
information which aids drug discovery process.
PATCH-CLAMP TCHNIQUE
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