Lecture 2

Question 1

How is voltage gated dependence of an ion channel shown

Answer 1

Whole cell patch clamp - As the membrane potential is increased by 20mV the open probability/ current increases more and more. If it weren’t a V dependent channel the current would remain the same upon increase of voltage.

Question 2

What is Ohms law and how can it be arranged for G

Answer 2

i = v/r       v=ir
G=1/R
R=1/G ----> V= I/G
G = I/V
G= I/V-Vrev

Question 3

How is an IV curve utilised to investigate the conductance/ potential relationship

Answer 3

Each point on the IV curve is calculated for G (G=I/V-Vrev) and plotted against V

Question 4

What shape does a depolarisation activated channel give

Answer 4

S-shaped curve.

Question 5

What is Vo

Answer 5

The voltage when G is 50% of its maximal value (Gmax)

Question 6

What is the Boltzman equation

Answer 6

A mathematical model for voltage dependence.

Question 7

How is Vo utilised in the investigation of WT and mutant channels

Answer 7

If a mutant channel causes a change in Vo, it can be concluded that the mutated amino acid must be important for the voltage-dependence of that ion channel.

Question 8

What is w

Answer 8

The gating charge - A measure of the movement of charge in the ion channel as it opens (due to charged amino acids in the pore region moving)

Question 9

What area of an ion channel is in control of voltage sensing

Answer 9

The S4 regions (4 of them) have different amino acid sequences, although some amino acids are highly conserved between the 4 regions

Question 10

What is the ball and chain mechanism of inactivation

Answer 10

A ball of amino acids are connected to the ion channel. When the ion channel goes from closed to open, the ball blocks the open pore and inactivates the channel. A closed state is different from an inactivated one and the channel can only be activated again once the channel has changed from its inactivated state to its closed one. This is why channels have a refractory period.

Question 11

What is the importance of the IFMQ3 mutant sodium channel

Answer 11

It has no ball and chain mechanism so fast inactivation of Na channels that would usually make it difficult to investigate activation is prevented.

Question 12

What does the “blip” on the graph represent

Answer 12

All the driving forces/ the clamped voltage becoming the same. Therefore any differences in current are due to differences in open probability.

Question 13

How can the blip be used to investigate mutations in the S4 regions of the IFMQ3 mutant Na channel

Answer 13

The voltage its originally clamped to and the current at the blip are plotted against eachother giving a sigmoidal curve representing the activation of the channel at different voltages. The Vo for different mutants can therefore be compared to the IFMQ3 mutant which has the same Vo as the WT channel.

Question 14

What was the result of the 1K4Q mutation of the Nav channel

Answer 14

Vo was much more positive - went from -29.1 mV in the IFMQ3 mutant to -10.6 in the 1K4Q mutant. This means in excitable cells, it is harder for action potentials to generate and propagate, dampening their excitability..

Question 15

Which charged amino acid in all of the domains (particularly 1st 2nd and 3rd are important for voltage dependence of the Nav channel

Answer 15

The 4th charged amino acid

Question 16

Where does the ball and chain lie in the Nav channels

Answer 16

In the III-IV linker region

Question 17

What was the result of mutating charged residues in 3s in the III-IV linker region

Answer 17

Had no effect on channel activation - charged residues therefore were deemed not important

Question 18

What was the result of deleting blocks of ten amino acids in the III-IV linker region. (blocks 2 and 3)

Answer 18

No current was produced in these mutated channels

Question 19

What was the result of deleting blocks of ten amino acids in the III-IV linker region. (blocks 1, 4 and 5)

Answer 19

The first and fourth group were the most important, loss of these groups led to a loss of fast inactivation.

Question 20

What is outward rectification

Answer 20

Outward positive currents are much larger than the inward currents

Question 21

What is ohmic rectification

Answer 21

No rectification occurring - linear relationship

Question 22

What is inward rectification

Answer 22

Much bigger inward (negative) current compared to a small outward one.

Question 23

What is Goldman rectification

Answer 23

Rectification completely independent of the channel - reliant on the solution the cell is in. If it is 140K inside the cell and 5K outside the cell then there will be a much larger movement of K ions out of the cell then into the cell. This is outward rectification. (assumes the open probability of the channel is not voltage dependent)

Question 24

Which residues in the ball and chain are responsible for closing the pore

Answer 24

The hydrophobic residues, specifically threonine (not hydrophobic bu still important), isoleucine, phenylalanine, methionine

Question 25

How does the Mg voltage dependent block cause inward rectification of the voltage independent Kir channel

Answer 25

When the potential is set at a voltage more negative than the reversal potential (when the current = 0) K enters the cell and causes an inward (negative) current. However when the potential is greater than the reversal potential Mg blocks the channel pore and an outward (positive) current cannot be generated.