Direct Measurement of Cardiac Na+ Channel Conformations Reveals Molecular Pathologies of Inherited Mutations.

Varga, Zoltan and Zhu, Wandi and Schubert, Angela R. and Pardieck, Jennifer L. and Krumholz, Arie and Hsu, Eric J. and Zaydman, Mark A. and Cui, Jianmin and Silva, Jonathan R. (2015) Direct Measurement of Cardiac Na+ Channel Conformations Reveals Molecular Pathologies of Inherited Mutations. Circulation. Arrhythmia and electrophysiology, 8 (5). pp. 1228-1239. ISSN 1941-3084

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Abstract

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.

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