Mechanisms of noncovalent β subunit regulation of NaV channel gating

Voltage-gated Na+ (Na-V) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound beta 1 and beta 3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the beta subunits affect the conformational dynamics of the cardiac Na-V channel (Na(V)1.5) voltage-sensing domains (VSDs). The pore-forming Na(V)1.5 alpha subunit contains four domains (DI-DIV), each with a VSD. Our results show that beta 1 regulates Na(V)1.5 by modulating the DIV-VSD, whereas beta 3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the beta 3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to beta 3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that beta 3 binds proximally to the DIII-VSD. Molecular-level differences in beta 1 and beta 3 interaction with the alpha subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting Na-V channel regulation of cell excitability.