Conformations of voltage-sensing domain III differentially define NaV channel closed- and open-state inactivation

Abstract

Voltage-gated Na+ (NaV) channels underlie the initiation and propagation of action potentials (APs). Rapid inactivation after NaV channel opening, known as open-state inactivation, plays a critical role in limiting the AP duration. However, NaV channel inactivation can also occur before opening, namely closed-state inactivation, to tune the cellular excitability. The voltage-sensing domain (VSD) within repeat IV (VSD-IV) of the pseudotetrameric NaV channel α-subunit is known to be a critical regulator of NaV channel inactivation. Yet, the two processes of open- and closed-state inactivation predominate at different voltage ranges and feature distinct kinetics. How inactivation occurs over these different ranges to give rise to the complexity of NaV channel dynamics is unclear. Past functional studies and recent cryo-electron microscopy structures, however, reveal significant inactivation regulation from other NaV channel components. In this Hypothesis paper, we propose that the VSD of NaV repeat III (VSD-III), together with VSD-IV, orchestrates the inactivation-state occupancy of NaV channels by modulating the affinity of the intracellular binding site of the IFMT motif on the III-IV linker. We review and outline substantial evidence that VSD-III activates in two distinct steps, with the intermediate and fully activated conformation regulating closed- and open-state inactivation state occupancy by altering the formation and affinity of the IFMT crevice. A role of VSD-III in determining inactivation-state occupancy and recovery from inactivation suggests a regulatory mechanism for the state-dependent block by small-molecule anti-arrhythmic and anesthetic therapies.

Figure 1.

The Na_V channel inactivation mechanism.(A) A schematic representation of mammalian Na_V channel shows four homologous repeats (I–IV) and the IFMT motif on the III–IV linker (yellow). (B) The structure of rNa_V1.5 (PDB accession no. 6UZ3) shows the IFMT motif enclosed by repeat III S5 and repeat IV S4–S5 linker and S5 and S6 segments, causing an allosteric block during inactivation.

Figure 2.

The models of open- and closed-state inactivation based on two VSD-III depolarized conformations.(A) During open-state inactivation, strong membrane depolarization leads to the activation of repeat I and II VSDs and the intermediate activation of VSD-III, resulting in the opening of the activation gate and the conduction of Na⁺ ions (I). Activation of the VSD-IV exposes the low-affinity binding site for the IFMT motif (II). Further translation of the VSD-III into its fully activated conformation establishes the stable inactivated configuration with high-affinity IFMT motif binding (III). (B) For closed-state inactivation at hyperpolarized membrane potential, VSD-III activates while repeats I and II VSDs are at rest, resulting in closed activation gate and no I_Na (I). Subsequent VSD-IV activation forms the low-affinity binding site for the IFMT motif (II). Since VSD-III occupies primarily in its intermediate conformation, the stable inactivated configuration is not established.

Figure 3.

A comparison between Na_VPaS and rNa_V1.5 structures. (A) Structures of eukaryotic Na_V channel reveal two distinct conformations of the depolarized VSD-III, varying in the number of gating charges (K and R) across the HCS. In Na_VPaS (left, light blue; PDB accession no. 5X0M), the activated VSD-III transfers two positively charged residues from an internal to an external side. The other VSD-III activated conformation from rNa_V1.5 (right, cyan; PDB accession no. 6UZ3) captures the total of four gating charges transfer. (B) An overlay of two Na_V channel structures shows that the VSD-III fully activated conformation (cyan) is needed to facilitate the binding of the IFMT motif.

Figure 4.

Experimental data supporting a two-step VSD-III activation model. (A) VCF recordings of rNa_V1.4 show simultaneous initiation of repeats I–III VSD activation (blue, red, and green) but a delay in VSD-IV activation (black), which is reduced with increasing voltages. Adapted from Chanda and Bezanilla (2002). (B) Fluorescence–voltage (FV) curves of hNa_V1.5 VCF constructs show that VSD-III activates at hyperpolarized potential, earlier than other repeat VSDs. Adapted from Varga et al. (2015). LFS, large fluorescence signal. (C) VCF recording of S1113C in rNa_V1.4 elicits two stages of VSD-III movement, with an initial increase followed by a decrease in fluorescence emission. Adapted from Chanda and Bezanilla (2002). (D) Coexpression of hNa_V1.5 and β3-subunit at high ratio (1:4 and 1:6) yields two distinct components of VSD-III activation kinetics, as detected by fluorescence emission (inset) and fluorescence–voltage curves. Adapted from Zhu et al. (2017). (E) A correlation between VSD-III deactivation time constants and the depolarization duration in WT hNa_V1.5 suggests the multiple steps of VSD-III activation, which is dependent on the IFMT motif as illustrated by the loss of correlation in IQM mutation. Adapted from Hsu et al. (2017). Error bars represent the standard error of the mean from the sample size of 3–6 measurements.