NaV Inactivation

Question 1

Why is inactivation of NaV important?

Answer 1

plays a crucial role in membrane excitability by contributing to the regulation of resting sodium channel availability

Question 2

Types of inactivation

Answer 2

1. Fast
2. Slow
3. Ultra Slow

Question 3

How does the voltage sensor relate to inactivation?

Answer 3

The time course of the activation of the voltage sensor is correlated with onset of inactivation and with the slow ON gating charge movement

Question 4

Is the DIV the inactivation gate?

Answer 4

Single-channel studies in an inactivation-deficient mutant showed that DIV is not the inactivation particle itself, but its movement causes a secondary conformational change in the pore (Goldschen-Ohm et al., 2013).

Question 5

How might DIV leading to a secondary change explain results from single-channel studies?

Answer 5

Slower opening presumably gives rise to the slow activation observed in single-channel studies (Aldrich et al.,1983)

Question 6

What did single channel studies show about NaV subconductance state?

Answer 6

Upon opening, Nav channels have an ~75% chance of entering the subconductance state, suggesting that the channels preferentially undergo transition from open to a subconductance state.

Question 7

Outline the domain movements in activation and inactivation

Answer 7

The activation of VSDI–III causes initial channel opening, whereas the subsequent activation of VSDIV uncovers a site for binding inactivation particle in the pore.

Question 8

What happens when the site for binding inactivation particle becomes exposed?

Answer 8

Inactivation follows rapidly once this site becomes available; therefore, the second opening is obscured in wild-type channels

Question 9

How do we disable DIV–S4 voltage sensing and what happens when this is done?

Answer 9

Disabling DIV–S4 voltage sensing by introduced glutamine residues at the first three charge-carrying residues slows entry into, and recovery from, fast inactivated states (Capes et al., 2013)

Question 10

How does fast inactivation occur?

Answer 10

A ‘hinged lid’ mechanism, in which a cytoplasmic region (the inactivating particle) occludes the pore by binding to a region nearby (the docking site)

Question 11

What does the inactivating particle contain?

Answer 11

A portion of the cytoplasmic linker connecting domains III and IV, with the crucial region centering on a four amino acid stretch consisting of isoleucine, phenylalanine, methionine and threonine (IFMT).

Question 12

What does the docking site contain?

Answer 12

multiple regions including the cytoplasmic linkers connecting segments 4 and 5 (S4-S5) in DIII and IV and the cytoplasmic end of the S6 segment in DIV

Question 13

What has been proposed about the movement of the voltage sensor in relation to the inactivating particle?

Answer 13

Miyamoto et al., 2001 proposed that the

1. outward movement of the voltage sensors (the S4 segments) exposes hydrophobic clusters in S4-S5
2. that can interact with the inactivating particle
3. after which the phenylalanine at position 1489 (F1489) interacts with:
   a) the alanine at position 1329 (A1329) in DIII S4-S5 and
   b) asparagine at position 1662 (N1662) in DIV S4-S5

Question 14

What are hairpin models of inactivation?

Answer 14

theoretical models of Sirota et al. (2002), who proposed that a hairpin motif optimizes the interaction between IFMT and its docking site, and that movement occurs around a previously identified hinge that is comprised of glycine (G) and proline (P) residues.