Much mechanistic insight into Na V channel gating has been recently provided by applying the voltage clamp fluorometry (VCF) protocol, which is used to fluorescently track VSD conformation and correlate VSD kinetics with ionic current gating. Both activation and inactivation gating have been previously shown to be modulated by the β1 and β3 subunits ( Morgan et al., 2000 Fahmi et al., 2001 Watanabe et al., 2009 Calhoun and Isom, 2014). Shortly thereafter, channels rapidly close-a process termed “fast inactivation” that is mediated by the intracellular DIII–DIV linker and the DIV-VSD ( West et al., 1992). Upon membrane depolarization, the S4 segments within the VSDs of DI–DIII are propelled outward to open the channel within a millisecond this is known as channel activation ( Chanda and Bezanilla, 2002).
S4, together with S1–S3, form the voltage-sensing domains (VSDs) and are coupled to the S5 and S6, which form the channel pore. The fourth segments (S4) contain multiple positively charged residues that move across the membrane in response to changes in membrane potential. Each domain is formed by six α helical transmembrane segments (S1–S6). The pore-forming Na V channel α subunit is composed of four homologous domains (DI–DIV) connected by cytoplasmic linkers ( Gellens et al., 1992). The dynamic expression patterns of β1 and β3 suggest that these two subunits play distinct roles in the regulation of Na V channel function and the action potential. β1 expression has been shown to increase ( Domínguez et al., 2005), whereas β3 has been shown to decrease through embryonic development ( Okata et al., 2016). Moreover, β1 and β3 also have a varied temporal expression profile during heart development. For example, the β1 and β3 subunits have been shown to differentially express in the atria and ventricles ( Fahmi et al., 2001 Watanabe et al., 2009 Yuan et al., 2014), suggesting that they may specifically tailor Na V channel function according to cell type. Intriguingly, even in the same organ, β subunit localization can differ ( Fahmi et al., 2001 Calhoun and Isom, 2014 Yuan et al., 2014). For instance, β1, but not β3, is highly expressed in skeletal muscles (The Human Protein Atlas). Despite the sequence homology between non-covalently associated β1 and β3 subunits, their expression profile across organs differs. The β subunits are widely expressed in many tissues, including the central and peripheral nervous system, the heart, and skeletal muscle ( Calhoun and Isom, 2014). Molecular-level differences in β1 and β3 interaction with the α subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting Na V channel regulation of cell excitability. Together, these results provide compelling evidence that β3 binds proximally to the DIII-VSD. Additionally, a fluorophore tethered to β3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Introduction of a quenching tryptophan into the extracellular region of the β3 transmembrane segment inverted the DIII-VSD fluorescence. Our results show that β1 regulates Na V1.5 by modulating the DIV-VSD, whereas β3 alters channel kinetics mainly through DIII-VSD interaction. The pore-forming Na V1.5 α subunit contains four domains (DI–DIV), each with a VSD. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the β subunits affect the conformational dynamics of the cardiac Na V channel (Na V1.5) voltage-sensing domains (VSDs). Key components are the non-covalently bound β1 and β3 subunits that regulate channel gating, expression, and pharmacology. Voltage-gated Na + (Na V) channels comprise a macromolecular complex whose components tailor channel function.