Patch clamp technique principle

patch clamp technique,types,principle.applications,limitations

1. Welcome to my Presentation

2. Presented by Dilruba Afrin Course : GEB-410 Dept. of Genetic Engineering & Biotechnology University of Rajshahi TITLE : Patch clamp technique

3. Contents Introduction What is patch clamping? Why used patch clamp? Basic principle Types and configurations Application Limitations

4. Patch clamp technique The patch clamp technique is a refinement of the voltage clamp. Erwin Neher and Bert Sakmann developed the patch clamp in the late 1970s and early 1980s . Neher and Sakmann received the Nobel Prize in Physiology or Medicine in 1991 for this work. The method used by Neher , Sakmann & Steinbach in1978 and subsequent refinements by Hamill ,Marty, Neher,Sakmann & Sigworth in 1981 have led to techniques for high resolution recording of current in excised membrane patches in addition to those that remain cell-attached. Introduction

5. Patch clamp technique Patch clamping is a widely applied electrophysiological technique for the study of ion channels; membrane proteins that regulate the flow of ions across cellular membranes and therefore influence the physiology of all cells. The technique can be applied to a wide variety of cells, but is especially useful in the study of excitable cells such as neurons, cardiomyocytes ,muscle fibers and pancreatic beta cells. It can also be applied to the study of bacterial ion channels in specially prepared giant spheroplasts . What is patch clamping?

6. Patch clamp technique 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 to measure current through ion channels vs. voltage,time,temperature,menthol,concentration etc. Patch clamp technique Why used patch clamp? Your Text Here

7. Patch clamp technique The patch-clamp technique allows the investigation of a small set or even single ion channels. It is thus of special interest in the research of excitable cells such as neurons, cardiomyocytes and muscle fibers. In principle, thin glass or quartz pipettes with a blunt end are sealed onto the membrane (Figure 2). Suction is applied to aid the development of a high-resistance seal in the gigaohm range. Basic principle of patch clamp

8. Patch clamp technique 3. This tight seal isolates the membrane patch electrically, which means that all ions fluxing the membrane patch flow into the pipette and are recorded by a chlorided silver electrode connected to a highly sensitive electronic amplifier. A bath electrode is used to set the zero level. To prevent alterations in the membrane potential, a compensating current that resembles the current that is flowing through the membrane is generated by the amplifier as a negative feedback mechanism (Figure 1). The membrane potential of the cell is measured and compared to the command potential. If there are differences between the command potential and the measurement, a current will be injected. Basic principle of patch clamp

9. Patch clamp technique This compensation current will be recorded and allows conclusions about the membrane conductance . The membrane potential can be manipulated independently of ionic currents and this allows investigation of the current-voltage relationships of membrane channels. Basic principle of patch clamp

10. Patch clamp technique Fig.1 : General principle of patch-clamp recordings. A glass pipette containing electrolyte solution is tightly sealed onto the cell membrane and thus isolates a membrane patch electrically. Currents fluxing through the channels in this patch hence flow into the pipette and can be recorded by an electrode that is connected to a highly sensitive differential amplifier. In the voltage-clamp configuration, a current is injected into the cell via a negative feedback loop to compensate changes in membrane potential. Recording this current allows conclusions about the membrane conductance.

11. Fig. 2: A phase contrast image of a patch pipette attached to the membrane of a cultured murine hippocampal neuron. Patch clamp technique

12. Patch clamp technique Several variations of the basic technique can be applied, depending on what the researcher wants to study – Cell-attached or on-cell patch Inside-out patch Whole-cell recording or whole-cell patch Outside-out patch Perforated patch Loose patch Automatic patch clamping Diagram showing configurations of the patch clamp technique Types and configurations

13. Patch clamp technique For the evaluation of antiarrhythmics agents. 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 molecular biology. Voltage clamp studies on sodium channels. Used to investigate a wide range of electrophysiological cell properties. Measurement of cell membrane conductance. Application

14. Patch clamp technique Imparting skillful training performance and recording in during single channel recording. Cost of process is expensive. Time consuming. Number of samples required is more at times. Chance of membrane distortion. Limitations

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