Lecture 15: Labarca et al., 1995

Question 1

What is a gate?

Answer 1

Barrier of ion flow in a channel

Question 2

What is gating?

Answer 2

Mechanism of how ion channels open and close

Question 3

What is the structure of a nicotinic AchR?

Answer 3

Four transmembrane regions, with a cytosolic loop between M3 and M4. C loop in extracellular binding domain will close in on the agonist.

Question 4

What is leucine 9’?

Answer 4

Leucine at the vertex that acts as the gate of the receptor

Question 5

What is the hydrophobicity around the L9’?

Answer 5

Hydrophobic residues in alignment around the L9’, there are hydrophilic residues right next to the gate –> maybe gating open leads to a favourable aa conformation.

Question 6

What is the oily knee model?

Answer 6

Unwin’s structure –> at the knee is the leucine (hydrophobic).

In the closed conformation, hydrophobic side chain is facing the pore
In the open conformation, L twists away from the pore, allowing ions to pass through

Question 7

What was the goal of Labarca’s study?

Answer 7

To explore the functional role of L 9’

Question 8

Where is L9’ located?

Answer 8

midway up the M2 helix in the muscle nAchR

Question 9

How did they analyze the function of L9’?

Answer 9

They used site directed mutagenesis

Question 10

What happened when they changed L9’ to a hydrophilic residue?

Answer 10

It altered the dose response curve so that less Ach was needed to get channel opening. 1000x lower concentration required to reach half max –> increase in sensitivity

Question 11

What occurred when more subunits were altered?

Answer 11

The concentration of Ach required to open the pore decreased as more subunits were mutated

Question 12

What does a Hill coefficient>1 mean?

Answer 12

That there is cooperativity in binding. The second binding Ach has a greated effect.

Question 13

What occurred to the Hill coefficient as more subunits were mutated?

Answer 13

The Hill coefficient decreased –> less Ach is required to open the channel

Question 14

How did the single channel recordings change after mutation of L9’?

Answer 14

In the mutant, there were more short openings, and the duration of openings was longer –> mutation allows channel to open for longer duration and makes the channel open more easily

Question 15

How did they use SCAM?

Answer 15

SCAM = substituted cysteine accessibility method

When MTSEA was added, it would cross-link with the cysteine that had replaced the leucine. When MTSEA was added, it cross-linked –> the side chain is facing the pore when activated by ACh

Question 16

What did the MTSEA experiments show?

Answer 16

That L9’ in closed conformation is not facing the pore, but when the channel is open, leucine is facing the pore. Also suggested that M2 is a beta strand structure –> every second residue faces the pore (is accessible by MTSEA)

Question 17

What are the interactions of L9’?

Answer 17

L251 makes side-to-side hydrophobic interactions with neighbouring alanine –> hydrophobic girdle
L251 and V255 form two hydrophobic rings, which would be the gate of the channel (highest constriction site)

Question 18

What happens to the structure of M2 during gating?

Answer 18

M2 straighten and move further apart during gating