Electrophysiological Techniques II

Question 1

Explain how Sakmann and Neher initially used the “patch clamp” technique to show individual ion channels opening in 1976.

Answer 1

• Isolated patch of membrane - sealed and electrically isolated from rest of cell
• Only one electrode needed
• ACh in patch pipette
• Observed small deflections in membrane current in the presence of ACh
• All deflections reached the same amplitude

Question 2

How did Sakmann and Neher improve the patch clamp technique in 1981?

Answer 2

GIGA-ohm (10⁹ ohm) seal was achieved by:

• Heat polishing the tip of the pipette
• Cleaning the cell membrane from debris such as connective tissue

The giga-ohm seal:

• Reduces background noise - can record very small currents - higher signal:noise ratio
• Increases mechanical stability of patch

Question 3

Describe the 4 different configurations of the patch clamp technique.

Answer 3

1. Cell-attached patch - mild suction - tight contact between pipette and membrane
2. Inside-out patch - patch taken out of membrane
3. Whole-cell recording - strong pulse of suction connects pipette interior with cytoplasm
4. Outside-out patch - extracellular domain of ion channel accessible

Question 4

Explain the advantages and disadvantages of the cell-attached patch.

Answer 4

Advantages:

• Agonists > pipette solution - ligand-gated ion channels
• Voltage steps > pipette - voltage-gated ion channels
• Agonists > extracellular solution - GPCRs

Disadvantages:

• RMP cannot be controlled
• Cannot control composition of intracellular medium

Question 5

Explain the advantages and disadvantages of the inside-out patch.

Answer 5

Advantages:

• Agonists > directly to cytoplasmic surface of cell membrane - e.g. activation of 2nd messenger systems
• Control composition of both extracellular and intracellular media

Disadvantages:

• Very reductionist approach - will channels behave normally?

Question 6

Explain the advantages and disadvantages of whole-cell recording.

Answer 6

Advantages:

• Agonists > pipette solution - e.g. 2nd messenger gated
• Agonists > extracellular solution
• Voltage steps > pipette
• Control composition of both extracellular and intracellular media

Disadvantages:

• Measures only macroscopic current - not individual channels opening
• Important intracellular components may dialysis out from the cell

Question 7

Explain the advantages and disadvantages of the outside-out patch.

Answer 7

Advantages:

• Can measure single channel openings
• Agonists > pipette solution
• Agonists > extracellular solution
• Voltage steps > pipette
• Control composition of extra/intracellular media

Disadvantages:

• Important intracellular components may be dialysis out from the cell