Patch clamp technique

1. PATCH CLAMP TECHNIQUE BY- RITIK VARDHAN M.Sc.(P) ROLL NO.- 1366

2. INTRODUCTION HISTORY PRINCIPLE CONFIGURATION & TYPES APPLICATION LIMITATION RECENT RESEARCH CONTENTS

3. 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 . 3 INTRODUCTION

4. 4 HISTORICAL DEVELOPMENT Graham

5. 5 Continues………………….

6. 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. 6 NEED OF PATCH CLAMP

7. THE PATCH-CLAMP TECHNIQUE Erwin Neher Bert Sakmann Germany (1991 Nobel Laureates) 7

8. 7/7/2011 8 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 standard deviation of the current noise at 1 kHz will be 0.04 pA, 8

9. The 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 G  allowing very small currents (10 -12 A) to be measured.

10. A patch-clamp rig

11. 11 CONFIGURATION OF PATCH CLAMP : On-cell Inside Out Whole Cell OutsideOut

12. Perforated patch Loose patch D Perforated-patch method (simplified) ASF TYPES

13. 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

14. Male wistar rat ISOLATION OF MYOCYTES

15. 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

16. 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

17. 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.

18. 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 .

19. 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

20. 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

21. 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

22. 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

23. 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

24. References Wyllie DJA (2007) Single-channel recording. In Patch-Clamp Analysis – Advanced Techniques 2 nd 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 Aidley DJ & Stanfield PR (1996) Ion Channels – Molecules in Action Cambridge University Press. This is a very good introductory textbook.