Mechanisms of noncovalent ß subunit regulation of NaV channel gating.

ABSTRACT

Voltage-gated Na+ (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound ß1 and ß3 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 ß subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). The pore-forming NaV1.5 α subunit contains four domains (DI-DIV), each with a VSD. Our results show that ß1 regulates NaV1.5 by modulating the DIV-VSD, whereas ß3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the ß3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to ß3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that ß3 binds proximally to the DIII-VSD. Molecular-level differences in ß1 and ß3 interaction with the α subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability.