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.