Voltage-gated Na+ (NaV) channels initiate excitation in neurons and myocytes, enabling rapid conduction over large distances that is decoupled from intracellular Ca2+ signaling (Hille, 2001). During excitation, NaV channel opening releases a large inward Na+ current (INa) that is followed by rapid inactivation, which occurs within milliseconds and makes most channels nonconductive. This inactivation from the open state is required to allow outward repolarizing currents to bring the cell membrane back to the resting potential. However, inactivation recovery is not instantaneous at hyperpolarized membrane potentials, nor is it limited to depolarized potentials. For example, in cardiac myocytes, NaV channels remain inactivated for 10s to 100s of milliseconds following action potential repolarization, rendering the myocyte refractory to excitation for a brief period and preventing reentrant arrhythmia (Zipes et al., 2017). In addition, neuronal memory of previous excitation can be conferred by inactivation of NaV channels that reduces the subsequent firing rate (Marom, 1998; Toib et al., 1998). At modest depolarized membrane potentials (less than −30 mV), NaV channels can also become inactivated before the activation gate opening but require longer depolarizations (Aldrich et al., 1983; Bean, 1981). This closed-state inactivation results in fewer available channels to initiate excitation. Thus, the regulation of NaV channel inactivation by time and membrane potential is essential for the functioning of excitable cells, allowing neurons and myocytes to appropriately respond to changes in membrane potential that span multiple time domains (Silva, 2014).

Mammalian NaV channels are formed by a single protein with four homologous repeats (I–IV; Fig. 1 A), each constituting six membrane-spanning segments (S1–S6). Within each repeat contains a voltage-sensing domain (VSD) that consists of segments S1–S4. The Na+-selective pore is formed jointly by the S5 and S6 segments (Yu and Catterall, 2003), with the region between S5 and S6 constituting the P-loop responsible for Na+ ion selectivity. Mammalian NaV channels additionally feature intracellular linkers and a unique C-terminal domain (CTD), which is known to bind various accessory subunits, including calmodulin and fibroblast growth factor homologous factors (Abriel, 2010). Given the complex time and voltage dependence of NaV channel inactivation, it must be connected to multiple different conformational states, whose occupancy is determined by the positions of the VSDs, the state of the channel pore, and the position of the CTD (Ulbricht, 2005).

Indeed, several reports have connected the activation of the VSD-IV to the onset of fast inactivation after channel opening (Capes et al., 2013; Goldschen-Ohm et al., 2013). Mutations in VSD-IV also suggest a role in closed-state inactivation (Kambouris et al., 2000; Chahine et al., 1994; Groome et al., 2011). Charge neutralization mutations within VSD-IV cause a large hyperpolarizing shift in voltage-dependent channel availability and a large fraction of inactivated channels at voltages where the channels are closed (Capes et al., 2013; Brake et al., 2021,Preprint). Early experiments demonstrated that the addition of intracellular pronase removed NaV channel inactivation and revealed the participation of intracellular components (Armstrong et al., 1973; Armstrong, 1981; Salgado et al., 1985). The model of NaV channel inactivation was then suggested to resemble the “ball-and-chain” model of K+ channel N-type inactivation, where a ball that is attached to the inner part of the channel causes inactivation by occluding the pore (Armstrong and Bezanilla, 1977; Hoshi et al., 1990). Subsequent experiments that disrupted the intracellular linker between repeats III and IV produced functional channels but lacked inactivation (Stühmer et al., 1989; Vassilev et al., 1988; Vassilev et al., 1989), further confining the sites of the inactivation gate to the III-IV linker. Finally, site-specific mutagenesis identified a hydrophobic cluster of amino acids, Ile-Phe-Met-Thr (IFMT), as an essential component for the inactivation mechanism (Hartmann et al., 1994), and a “hinged-lid” model was proposed (Eaholtz et al., 1994; Kellenberger et al., 1997; Rohl et al., 1999; West et al., 1992). In this model, the loop between two hinged points serves as a rigid lid that folds over the channel pore, with the IFMT motif acting as a hydrophobic latch to stabilize the inactivated conformation. Additional mutagenesis and the discovery of inherited proarrhythmic mutations within cardiac NaV channels further suggested that the binding site or the “receptor” for the IFMT motif involves repeats III and IV S4–S5 linkers and IV S6 segment (Smith and Goldin, 1997; McPhee et al., 1995; McPhee et al., 1998). Recent structures of eukaryotic NaV channels, however, reveal that the IFMT motif resembles more of a wedge that squeezes into the crevice formed by repeat III S5 and repeat IV S4–S5 linker and S5 and S6 segments (Yan et al., 2017; Pan et al., 2018, 2019; Shen et al., 2019; Jiang et al., 2020; Li et al., 2021) and allosterically blocks the NaV channel conduction pore (Fig. 1 B).

Apart from the inactivation gate, the VSD-III, the CTD, and the state of the channel pore have also been implicated in NaV channel inactivation (Cha et al., 1999; Deschênes et al., 2001; Hsu et al., 2017; Mantegazza et al., 2001; Motoike et al., 2004; Pitt and Lee, 2016, Mangold et al., 2017). Based on new structural data in combination with numerous previous functional studies, we hypothesize that the activation of the VSD-III, in addition to the VSD-IV, facilitates different inactivated states by modulating the binding affinity of IFMT crevice. In this Hypothesis paper, we will first outline the models of each inactivated state. Then, we will present the structural and electrophysiological evidence that supports our hypothesis and leads to the proposed model. Finally, we will discuss the model implications on the modulation of NaV channel function by accessory subunits and therapeutic drugs.