Action potential generation is controlled by the opening, inactivation,

and recovery of voltage-gated sodium channels (NaV). A-type fibroblast

growth factor homologous factors (A-type FHFs), which are cytosolic

NaV binding proteins, mediate rapid-onset, long-term inactivation (LTI)

of NaV by contributing an independent inactivation particle that

competes with the NaV intrinsic fast inactivation mechanism (Dover et

al., J. Physiol. 588:3695; 2010). To further explore the structural

mechanism of A-type FHF induced LTI of NaV, we have performed

cotransfection of wild-type and mutant FHF2A expression plasmids

together with wild-type and mutagenized TTX-resistant Nav1.6 and

Nav1.5 expression plasmids into Neuro2A cells, and have analyzed

TTX-resistant sodium currents through various whole cell voltage

clamp protocols. Our results demonstrate that LTI induced by FHF2A

only occurs when the channels reach the open state. FHF2A can

compete for binding to channels with another open channel blocking

protein, NaVb4, which is responsible for resurgent sodium current due to

its rapid dissociation from channel upon repolarization. Both FHF2A

and NaVb4 blocking particles rely upon hydrophobic and basic residues,

suggesting they may share a common binding surface within NaV's

cytoplasmic cavern exposed in the open state. However, FHF2A

dissociates much more slowly from channels than does NaVb4,

accounting for their functional dissimilarity. Ongoing channel

mutagenesis seeks to reveal channel binding sites for each of these

blocking particles. In summary, our studies show that A-type FHFs

serve as open sodium channel blockers, in competition with both the

NaV intrinsic fast inactivation mechanism and the accessory subunit

NaVb4, to drive channels into a long-term refractory state.

Experimental Approach

A-Type Fibroblast Growth Factor Homologous Factors Function as

Open Channel Blockers to Modulate Voltage-gated Sodium Channel Availability Yue Liu1,2, Mitchell Goldfarb1

1Department of Biological Sciences, Hunter College of City University, 695 Park Avenue, New York, NY 10065, USA 2The Graduate Center Biology Program, City University of New York, New York, NY 10016, USA

Voltage-gated sodium channels (NaV) consist of a large a subunit,

the core of the channel which can function on its own. The a subunit

has four domains (I~IV), each containing six membrane-spanning

regions (S1~S6).

NaV Mediates inward current of action potentials upon membrane

depolarization of excitable cells. NaV inactivation can be caused by

its intrinsic fast inactivation particle and accessory proteins (A-type

FHFs and NaVb4).

Here we study long-term inactivation of NaVa induced by FHF2A

(one form of A-type FHFs) and the functional competition between

these two accessory protein.

Abstract

Results

Acknowledgement Supported by NIH/NIGMS R01-GM095430

Grateful to all members in Prof. Mitchell Goldfarb’s lab

Introduction

N-terminal hydrophobic and basic FHF2A residues

mediate sodium channel long-term inactivation

(A) N-terminal sequence of FHF2A and engineered FHF2A mutations.

(B) Nenro2A cells transfected with NaV1.6 +/- FHF2A and its mutants

were subjected to 4 pulses of depolarization separated by 40 msec -90

mV intervals. Examples of mutants that reduce or eliminate long-term

inactivation (LTI) accumulation.

(C) Mutations of

hydrophobic (upper panel)

and basic (lower panel)

amino acids in the N-

terminal FHF2A reduce or

eliminate LTI accumulation.

All FHF2A mutants that reduce long-term

inactivation accelerate sodium channel recovery

(FHF2A dissociation) rate

All FHF2A mutants that reduce long-term inactivation accelerate sodium channel recovery (FHF2A dissociation) rate All FHF2A mutants that reduce long-term inactivation accelerate sodium channel recovery (FHF2A dissociation) rate

(A) Long-term recovery of NaV1.6 in the cells co-expressing FHF2A (WT)

(B) FHF2A (R11Q/R17Q)

dissociation from NaV1.6.

FHF2A long-term inactivation particle is an

open-channel blocker

(A) Neuro2A cells transfected with NaV1.6

(F1478Q) + FHF2A were depolarized to

different test voltages to assay channel

activation, each depolarization was

followed by 40 msec -90 mV recovery and

a second depolarization to 0 mV; current

induced by second depolarization reported

LTI induced by test voltage. LTI induction

required activation of some channels,

indicating open-channel block.

(B) Voltage

dependence of

channel activation and

LTI are very similar

Summary

FHF2A long-term inactivation and NaVb4

blocking particles engage in reciprocal functional

competition

(A) Neuro2A cells expressing fast inactivation defective NaV1.5 (F1486Q)

were depolarized with 4 pulses of 48 msec +70 mV separated by 40 msec

-100 mV intervals (ENa≈-10 mV). F2A (2-18) peptide drives the channels

into maximal LTI after the first pulse (upper-right). Navβ4 (154-167) peptide

blocks the channels at +70 mV but dissociates rapidly upon repolarization

to -100 mV, giving 4 cycles of resurgent currents (lower-left). With both F2A

and b4 peptides, the channels undergo accumulated LTI, with gradually

decreasing resurgent current (lower-right).

(B) LTI of NaV1.5 (F1486Q)

induced by F2A +/- β4

(C) Resurgent current

by β4+/- F2A

1.A-type FHF long-term

inactivation (LTI) particle

functions as open-channel

blocker.

2.A-type FHFs competes

with another open-

channel blocker Navβ4

functionally to modulate

NaV availability.

3.N-terminal hydrophobic

and basic residues of A-

type FHFs contribute to

NaV LTI.

(C) Recovery of NaV1.6 in the cells

expressing different FHF2A mutants.