Methods to study ion channels

Question 1

What are ion channels ?

What purpose do they serve ?

Answer 1

Ion channels are the gateway for the exchange of charge between cells and the extracellular
environment
Salts readily dissociate in water and their ions are associated with water molecules
Ion channels provide the water environment for the passage of ions across the membrane, while the lipid
bilayer contains no water and no charges and is therefore
impermeable to ions

Question 2

What are the 4 main types of ion channels ?

What are their main characteristics >

Answer 2

1. LGICs : typically found at synapses
2. Second messenger operated channels : typically found at pre or post synaptic terminals, but also away from synapses
3. VGCs : found everywhere on the soma, axon and dendrites of neurons (closed at resting potential, open upon depolarization)
4. Stretch activated channels : found in mechanosensors, but also on many cell membranes (detect membrane tension)

Question 3

How does the conductance of the membrane change with an increasing number of open channels ?

Answer 3

Conductance increase because the channels are inserted in parallel.

Question 4

What is the usual range of conductance of a single ion channel ?

Answer 4

1-100pS (a few fall outside this range)

Question 5

What does patch clamp allow ?

What was used prior to this ?

Answer 5

Patch clamp allows the measure electrical signals on a tiny patch of membrane as well as through the whole cell.
Previously, sharp electrodes were used to impale the cell.
These could only measure the voltage difference between inside and outside the membrane

Question 6

How does patch clamp differ from recording with sharps ?

Answer 6

Patch electrodes are blunt and stuck on the membrane
Microlectrodes are sharp and stuck inside the cell. Both patch and sharp electrodes can measure the membrane voltage, but patch electrodes can do this only in “whole cell” mode.

Question 7

How does patch clamping work ?

Answer 7

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

Question 8

Describe the 4 patch clamp configurations.

Answer 8

1. Cell attached : We can record the activity of all the channels contained in the small patch of membrane within the pipette. 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
2. Inside-out : The inside of the membrane is accessible. It is therefore useful for looking at the effects of internal metabolites on channels
3. Outside-out : 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
4. Whole-cell : For measuring the contribution of channels to the total membrane current. The electrode sees all the channels in the membrane. 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

What does voltage clamp allow ?

Answer 9

The measurement of voltage and time-dependant changes in conductance.

Question 10

What is an idealized Vclamp similar to in a circuit ?

How is this Vclamp connected to the cell ?

Answer 10

To a battery holding the voltage constant across the membrane.
The “battery” is connected to the cell through the patch electrode.

Question 11

How does voltage clamp work ?

Answer 11

In a real voltage clamp circuit a feedback amplifier compares the voltage across the membrane with the imposed command voltage and injects the current needed to make this difference 0.

Question 12

Total current = I = NiP
g = i/V
Define all the terms of these equations.

Answer 12

i = current that flows through a single channel when it opens (single channel currents are small (~0.1-100 pA), but 2-5 pA are the most common)
g = single channel conductance (conductances are between 1-100 pS)
P = fraction of time the channel spends in the open state (it can be very brief, form a few us to a few ms, and is normally dependent on voltage)
i is the charge movement per second. The rate of ion movement across a channel is typically of 1-50e7 ions per second (c.f fastest enzymes catalyze 10e5 reactions per second can)
N =number of channels (for our purposes N is the number of channels within the pipette tip area)

Question 13

How will the IV curves of voltage dependent Vs voltage independent curves look like ?

Answer 13

Voltage dependent channel ==> non-linear curve

Voltage independent curve ==> linear curve

Question 14

What are the 3 states that v-gated channels can be found in ?

Answer 14

Closed channel (favored at -ve potentials)
Open channel (favored at +ve potentials)
Inactive channel
Open --> Closed --> Inactive --> Open etc...

Question 15

Which states can a ligand gated ion channel enter when it is exposed to a short agonist application ?
What about during a long agonist application ?

Answer 15

Short : R –> AR –> AR*

Long : R –> AR –> AR* –> DR