Post on 22-Jan-2018
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.