PATCH CLAMP TECHNIQUE UNDER THE GUIDENCE OF MISS MEENU SINGH ASST.PROFEESOR DEPARTMENT OFPHARMACOLOGY CMRCP BY KRISHNAPRIYA.P M.PHARM PHARMACOLOGY
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 in 1991 .
HISTORICAL DEVELOPMENT Graham
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.
THE PATCH-CLAMP TECHNIQUE Erwin Neher Bert Sakmann Germany (1991 Nobel Laureates)
BASIC PRINCIPLE 7/7/2011 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 ﬁ re -polished glass pipette Electrolyte solution Electrode (10-25 µm) Renitence S i 2 = 4kTf c /R variance of the current noise (in A 2 ) k = Boltzmann’s constant, T = temperature (° Kelvin), fc = the bandwidth(Hz) <10nm 10 GΩ resistor at 20°C, the standard deviation of the current noise at 1 kHz will be 0.04 pA,
TYPES OF PATCH CLAMP :
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
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
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
Requires strong background inc channel biophysics
Imparting skillful training performance and recording In during single channel recordings
Cost of process is expensive
Number of samples required is more at times
Chance of membrane distortion
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.
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
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.