Abstract
Voltage-gated Na+ (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound β1 and β3 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 β 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 β1 regulates NaV1.5 by modulating the DIV-VSD, whereas β3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the β3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to β3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that β3 binds proximally to the DIII-VSD. Molecular-level differences in β1 and β3 interaction with the α subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability.
Highlights
In electrically excitable organs, such as the heart, brain, and skeletal muscle, voltage-gated Na+ (NaV) channels cause the initiation and propagation of action potentials by conducting a large and rapid inward Na+ flux
This result suggests that β1 and β3’s modulation of the DIV-voltage-sensing domains (VSDs) is not additive. These results show that β1 and β3 binding to the NaV1.5 channel is not exclusive, and when both β subunits are present, their cooperativity further depolarizes DIII-VSD activation, which leads to additional modifications of channel activation
We discovered that WT β1 subunit coexpression shifts DIV-VSD activation to depolarized potentials, consistent with the shift in steady-state inactivation (SSI). β1 subunits relieve the immobilization of the DIII and DIV VSDs by fast inactivation, which potentially contributes to the increased rate of channel recovery from inactivation induced by β1
Summary
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 NaV channel (NaV1.5) voltage-sensing domains (VSDs). Our results show that β1 regulates NaV1.5 by modulating the DIV-VSD, whereas β3 alters channel kinetics mainly through DIII-VSD interaction. A fluorophore tethered to β3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that β3 binds proximally to the DIII-VSD. Molecular-level differences in β1 and β3 interaction with the α subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability
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