Figure 2.

Functional characterization of the variants in the FHF2A N-terminal domain

(A) Scheme of voltage-dependent sodium (Na_v) channel state transitions as modulated by A-type fibroblast growth factor homologous factors (FHFs), as described previously.^31,33 Strong rapid depolarization results in transient opening of channels and sodium influx (vertical red arrow). Within milliseconds, channels intrinsically inactivate (leftward descending arrow). When channels are physically associated with A-type FHF, the N terminus (red triangle) competes with the intrinsic Na_v mechanism (blue circle) to rapidly induce long-term inactivation (rightward descending arrow). Recovery to the closed state upon repolarization requires hundreds of milliseconds. Long-term inactivation of channels is impaired in presence of the FHF2A_R11C or FHF2A_R14T (white asterisk). A-type FHFs raise the voltage dependence of steady-state inactivation, preserving more channels in the closed (resting) state. The pro-excitatory DEE-associated FHF1A_R114H mutant protein further raises the voltage dependence of steady-state inactivation.⁴

(B) Voltage-clamp protocol for accumulating long-term inactivation used in (C)–(E). The 40 ms intervals at −90 mV allow for full recovery from intrinsic fast inactivation, but only partial recovery from long-term inactivation, which has a far slower recovery rate.

(C–E) Representative sodium current traces in Neuro2A cells expressing Na_v1.6 together with either (C) FHF2A_WT, (D) FHF2A_R11C, or (E) FHF2A_R14T. The variants impair channel long-term inactivation.

(F) Analysis of long-term inactivation induced by wild-type and mutant FHF2A proteins. n represents the number of transfected cells recorded for a given experimental protocol. Data points represent the mean ± standard error (error bars) from the n recorded cells. The impaired long-term inactivation of FHF2A_R11C and FHF2A_R14T in comparison to FHF2A_WT upon depolarizations 2,3,4 is highly significant (p < 10⁻⁷).

(G) Analysis of Na_v1.6 recovery from long-term inactivation at −90 mV. Na_v1.6 recovers faster in cells expressing FHF2A_R11C or FHF2A_R14T compared to cells expressing FHF2A_WT.

(H) FHF2A_R11C and FHF2A_R14T retain the ability to induce 17–19 mV depolarizing shifts in the voltage dependence of Na_v1.6 steady-state inactivation.