BACKGROUND: -Dysregulation of voltage-gated cardiac Na+ channels (NaV1.5) by inherited
mutations, disease-linked remodeling, and drugs causes arrhythmias. The molecular
mechanisms whereby the NaV1.5 voltage-sensing domains (VSDs) are perturbed to pathologically
or therapeutically modulate Na+ current (INa) have not been specified. Our aim was
to correlate INa kinetics with conformational changes within the four (DI-DIV) VSDs
to define molecular mechanisms of NaV1.5 modulation. METHOD AND RESULTS: -Four NaV1.5
constructs were created to track the voltage-dependent kinetics of conformational
changes within each VSD, using voltage-clamp fluorometry (VCF). Each VSD displayed
unique kinetics, consistent with distinct roles in determining INa. In particular,
DIII-VSD deactivation kinetics were modulated by depolarizing pulses with durations
in the intermediate time domain that modulates late INa. We then used the DII-VSD
construct to probe the molecular pathology of two Brugada Syndrome (BrS) mutations
(A735V and G752R). A735V shifted DII-VSD voltage-dependence to depolarized potentials,
while G752R significantly slowed DII-VSD kinetics. Both mutations slowed INa activation,
even though DII-VSD activation occurred at higher potentials (A735V) or at later times
(G752R) than ionic current activation, indicating that the DII-VSD allosterically
regulates the rate of INa activation and myocyte excitability. CONCLUSIONS: -Our results
reveal novel mechanisms whereby the NaV1.5 VSDs regulate its activation and inactivation.
The ability to distinguish distinct molecular mechanisms of proximal BrS mutations
demonstrates the potential of these methods to reveal how inherited mutations, post-translational
modifications and anti-arrhythmic drugs alter NaV1.5 at the molecular level.
