Clamping

Question 1

How does the patch clamp work?

Answer 1

The glass sticks to the lipid membrane and
form a Gigaseal, a seal with resistance in
the order of several G!. Virtually no
current can flow between the membrane
and the glass .

Question 2

What are the 4 configurations of patch clamp?

Answer 2

Cell-attached, inside-out, outside-out and whole cell

Question 3

When would I use a cell-attached configuration?

Answer 3

When you want to record the activity of all the

channels contained in the small patch of membrane within the pipette.

Question 4

How would I adjust my cell-attached configuration for different types of channels?

Answer 4

If the channels are voltage activated we can change the

holding voltage. If they are ligand gated, we can include the agonist in the pipette solution

Question 5

When would I use the inside-out configuration?

Answer 5

looking at the effects of internal metabolites on channels

Question 6

When would I use the outside-out configuration?

Answer 6

The outer side of the membrane is exposed. It is therefore useful for looking at the effects of external ligands, typically transmitters or antagonists, on channels function. It is often used for ‘concentration jumps’, that are ‘synaptic like’ fast application of transmitter

Question 7

When would I use whole cell configuration?

Answer 7

For measuring the contribution of channels to the total
membrane current. The electrode sees all the
channels in the membrane.

Question 8

Benefits and drawbacks of whole cell configuration.

Answer 8

The cytoplasm of the cell diffuses out and is replaced by
the pipette solution. It gives control of ion concentrations,
at expense of loss of metabolites important for
channel function or modulation

Question 9

Voltage clamp allows measurements of

Answer 9

voltage and time dependent changes in conductance

Question 10

What does the feedback amplifier in the voltage clamp do?

Answer 10

Compares the voltage across the membrane with the imposed command voltage and injects the current needed to make this difference =0

Question 11

Closed channel is favoured at

Answer 11

negative potentials

Question 12

Open channel is favoured at

Answer 12

positive potentials

Question 13

How can we separate the different current components?

Answer 13

• By modifying the concentration gradient of one of the
  critical ions (i.e. Na+);
• By substituting one of the critical ions (i.e. Na+) with
  an impermeant one;
• By selective pharmacological blockade of the
  different voltage-sensitive channels (i.e. TTX for Na+
  channels, TEA for K+ channels)