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Patch clamp ppt by kp
 

Patch clamp ppt by kp

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    Patch clamp ppt by kp Patch clamp ppt by kp Presentation Transcript

    • PATCH CLAMP TECHNIQUE 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 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 :
      • On-cell
      • Inside Out
      • Whole Cell
      • OutsideOut
    • TYPES
      • Perforated patch
      • Loose patch
      • Blind patch
    • 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
      • .
    • 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