Patch clamp technique
- 1. Name- Sonika Kumari Roll no.- 201650 Central University of Haryana PATCH CLAMP TECHNIQUE
- 2. • Patch Clamp Technique developed by Erwin Neher and Bert Sakmann in 1976, • very small currents are measured through a tiny region of the membrane surface containing only one or a few ion-channel molecules. • measurement the size and duration of the current that flows during one opening of an ion channel. • determine how often a channel opens and how that frequency is affected by membrane potential, regulatory ligands, toxins, and other agents. • Patch-clamp studies have revealed that as many as 104 ions can move through a single ion channel in 1 ms. https://personalizeaf.net/wp-content/uploads/2021/04/Screenshot-2021-04-06-at-17.24.58.png Introduction
- 3. • Such an ion flux represents a huge amplification of the initial signal; for example, only two acetylcholine molecules are needed to open an acetylcholine receptor channel. • Because a single ion channel typically remains open for only a few milliseconds, monitoring this process is beyond the limit of most biochemical measurements.
- 4. A finely drawn-out pipette (micropipette) is pressed against the cell surface, and negative pressure in the pipette forms a pressure seal between pipette and membrane. As the pipette is pulled away from the cell, it pulls off a tiny patch of membrane (which may contain one or a few ion channels). After placing the pipette and attached patch in an aqueous solution, channel activity as the electric current that flows between the contents of the pipette and the aqueous solution can be measured. In practice, a circuit is set up that “clamps” the transmembrane potential at a given value and measures the current that must flow to maintain this voltage. Electrical measurements of ion-channel function
- 5. Source- Lehninger Principle of Biochemistry, 6th Edition
- 6. .With highly sensitive current detectors, researchers can measure the current flowing through a single ion channel, typically a few picoamperes. . The trace shows the current through a single acetylcholine receptor channel as a function of time (in milliseconds), revealing how fast the channel opens and closes, how frequently it opens, and how long it stays open. . Downward deflection represents channel opening. Clamping the Vm at different values permits determination of the effect of membrane potential on these parameters of channel function.
- 7. For evaluation of anti arrhythmic agents To study a cardio selective inhibition of ATP sensitive potassium channel To identify multiple types of calcium channels To measure the effect of potassium channel openers Voltage clamp studies on sodium channels To investigate wide range of electro-physiological cell properties Measurement of cell membrane conductance Applications
- 8. • Cost of process is very expensive • Time consuming • Number of samples required are more at a time • Chance of membrane distortion • Imparting skilful training and recording in during single channel recording. Limitations Patch-clamp is highly modified and successful technique Development of this technique is being done for newer approaches to yield accurate and efficient information which aids drug discovery process
- 9. References- • Lehninger Principles of Biochemistry, 6th Edition