Patch clamp techniques

PATCH CLAMP TECHNIQUES

1. Patch Clamp Technique By Ved prakash panda M.Pharm (Pharmacology)

2. DEFINITION The patch clamp technique is a laboratory technique in electrophysiology that allows the study of single or multiple ion channels in the cells. This technique is the refinement of the voltage clamp.

3. NEED OF PATCH CLAMP Measures current through ion channels. Provides access to the inside of the cell 1. Can insert an electrode into the cell 2. Can change intracellular fluid. Provides for low noise recordings of current.

4. APPLIES ON Neurons Cardiac myocytes Muscle fibre Pancreatic beta cells Kidney cells

5. APPLICATIONS To identify multiple types of calcium channels To study electrophysiological cell properties Measurement of cell membrane conductance For evaluation of anti arrythmic agents

6. PRINCIPLE

7. In this method a patch of membrane is isolated electrically from the external solution and record current flowing into the patch. This is done by pressing a fire polished glass pipette which is filled with suitable electrolyte solution by applying light suction. 10G ohm resistor at 20 degrees Noise- 1kHz

8. PATCH CLAMP CIRCUIT Resistance 50Gohm Current 10^-12 amp

9. `VARIATIONS OF PATCH CLAMP : Cell attached clamp Inside out patch Whole cell patch Outside out patch Perforated patch Loose patch

10. CELL ATTACHED PATCH Allows recording of the current through single or few ion channels contained in the patch of the membrane

11. INSIDE OUT PATCH It has the access to the intracellular surface of the membrane via the bath and can change the chemical composition of what the surface of the membrane is exposed to.

12. WHOLE CELL PATCH Larger opening at the tip of the patch clamp electrode provides lower resistance and thus better electrical access to the inside of the cell. Because the volume of electrode is larger than volume of the cell , the contents of cell will be replaced by contents of electrode

13. OUTSIDE OUT PATCH

14. This process involves more steps and results in lower frequency of usable patches. Places the external rather than intracellular surface of the cell membrane on the outside of the path of membrane in relation to the patch electrode.

15. PERFORATED PATCH Suction is not used to rupture the patch membrane. The electrode solution contains small amounts of anti fungal or antibiotic agents which diffuses into the patch and forms small pores in the membrane.

16. LOOSE PATCH Employs a loose seal(lower electrical resistance) The pipette is moved slowly towards the cell until the electrical resistance of the contact between the cell and pipette increases a few times greater resistance than that of the electrode.

17. WHOLE CELL CONFIGURATION

18. PATCH CLAMP TECHNIQUE IN ISOLATED CARDIAC MYOCYTES

19. PATCH CLAMP TECHNIQUE IN KIDNEY CELLS It is used to study the role of each nephron part in urine production and the mechanism of transportation of substances in the tubular cell membrane. PRINCIPLE: In the different parts of the kidney fluid is reabsorbed and the substances may be transported either from the tubular lumen to the blood side (reabsorption) and vice versa (secretion)

20. PROCEDURE Freshly isolated kidney cells of rabbit are isolated. Segments of the nephron are dissected and perfused with one end with perfusion system. The non cannulated end is attached with a patch pipette The patch pipette is kept in contact with the brush border membrane After the slight section of the patch electrode single potassium or sodium channels reading scan be recorded.

21. PATCH CLAMP TECHNIQUE IN BETA CELLS Beta cells in the pancreatic islets of langerhans are the biological sensors for glucose and plays a major role in balancing the catabolic and anabolic needs. Beta cell response to glucose are oscillatory changes of membrane potential ate tightly coupled with oscillatory changes in intracellular calcium concentration. Both membrane potential and calcium changes spreads from one cell to another in a wave like manner.

22. PATCH CLAMP TECHNIQUE IN CANCER CELLS Voltage gated ionic channels are known to be involved in oncogenesis . Breast cells proliferate and migrate under the constant activation of growth factors , hormones The activity of ionic channels is modulated by pathways such as kinases , phosphatases which in turn effect oncogenic properties.

23. PROCEDURE The electrophysiological properties of the sodium channel in the breast cancer cell line MDA-MB 231 is known. With perforated patch a configuration which allows to keep the cytoplasm intact the mean current amplitude was lower, the relative conductance voltage relationship was shifted to more positive and from the recovery from the inactivation was accelerated when compared to ruptured patch. The results involving kinases or phosphatases are switched off when the cytoplasm is diluted.

24. Thank you