Jgp_201611678 389..404

In neurons and myocytes, voltage-gated Na (NaV) channels initiate membrane excitation. Shortly thereafter, fast inactivation diminishes the Na current, which allows K efflux to return the membrane to its resting potential. Inactivation kinetics cannot be described by a single time constant, but instead require a collection of relaxations whose time domains span from milliseconds to minutes (Silva, 2014; Marom, 2016). After myocyte and neuron membranes return to their resting potential, the rate of recovery from NaV channel inactivation determines the period during which they are refractory to excitation. In addition to the conduction velocity of excitation, the refractory period plays a key role in determining whether the myocardium is able to sustain deadly arrhythmic activity (Mines, 1913). Much effort has been put into mapping inactivation to the eukaryotic NaV channel structure. Functional Na channels are formed by monomers with four homologous domains (DI–DIV), each with six transmembrane-spanning segments (S1–S6). DI–DIV are each composed of a voltage-sensing domain (VSD) formed by S1–S4 and a pore domain that includes S5–S6. Within the VSD, a charged segment (S4) responds to changes in membrane potential to activate and deactivate the channel (Catterall et al., 2005; Ahern et al., 2016). Inactivation is associated with a hydrophobic amino acid sequence (Ile-Phe-Met-Thr) known as the IFM or IFMT motif that is located within the DIII–DIV linker (Vassilev et al., 1988; Stühmer et al., 1989; West et al., 1992). Inactivation is also significantly modulated by the S4–S5 linkers of DIII (Smith and Goldin, 1997) and DIV (McPhee et al., 1998), stretches of the DI-S6 and DIV-S6 segments (McPhee et al., 1994, 1995; Wang et al., 2003), and the C terminus (Deschênes et al., 2001). Insight into the role that the VSDs play in modulating inactivation has been gained by tethering fluorophores to the extracellular S4 segments (Chanda and Bezanilla, 2002; Chanda et al., 2004), which allows correlation of changes in VSD conformation to ionic current kinetics, including inactivation (Cha et al., 1999; Silva and Goldstein, 2013a,b). Recent application of this method, known as voltage-clamp fluorometry (VCF), has shown that DIV-VSD conformation is tightly connected to the rapid components of inactivation in the rat skeletal muscle Na channels, rNaV1.4 (Capes et al., 2013). The DIII-VSD of rNaV1.4 has been shown to modulate channel opening (Muroi et al., 2010) and interact with pore-binding local anesthetics (Sheets and Hanck, 2003; Muroi and Chanda, 2009; Arcisio-Miranda et al., 2010), but its inactivation role is less well defined. In the human cardiac NaV channel, hNaV1.5, inactivation defects predispose patients to arrhythmia and sudden death. In long QT syndrome type 3 patients, hNaV1.5 inactivation is often impaired, and the increase in persistent Na current causes prolonged action potentials and triggered activity. In contrast, Brugada syndrome mutations often enhance hNaV1.5 inactivation, which reduces peak Na current and causes pro-arrhythmic conduction abnormalities (Silva and Silva, 2012; Havakuk Functional eukaryotic voltage-gated Na (NaV) channels comprise four domains (DI–DIV), each containing six membrane-spanning segments (S1–S6). Voltage sensing is accomplished by the first four membrane-spanning segments (S1–S4), which together form a voltage-sensing domain (VSD). A critical NaV channel gating process, inactivation, has previously been linked to activation of the VSDs in DIII and DIV. Here, we probe this interaction by using voltage-clamp fluorometry to observe VSD kinetics in the presence of mutations at locations that have been shown to impair NaV channel inactivation. These locations include the DIII–DIV linker, the DIII S4–S5 linker, and the DIV S4-S5 linker. Our results show that, within the 10-ms timeframe of fast inactivation, the DIVVSD is the primary regulator of inactivation. However, after longer 100-ms pulses, the DIII–DIV linker slows DIII-VSD deactivation, and the rate of DIII deactivation correlates strongly with the rate of recovery from inactivation. Our results imply that, over the course of an action potential, DIV-VSDs regulate the onset of fast inactivation while DIII-VSDs determine its recovery. Regulation of Na channel inactivation by the DIII and DIV voltagesensing domains