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

Zoltan Varga, Wandi Zhu, Angela R. Schubert, Jennifer L. Pardieck, Arie Krumholz, Eric J. Hsu, Mark A. Zaydman, Jianmin Cui, Jonathan R. Silva

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

Supplemental Digital Content is available in the text. Background - Dysregulation of voltage-gated cardiac Na + channels (Na V 1.5) by inherited mutations, disease-linked remodeling, and drugs causes arrhythmias. The molecular mechanisms whereby the Na V 1.5 voltage-sensing domains (VSDs) are perturbed to pathologically or therapeutically modulate Na + current (I Na) have not been specified. Our aim was to correlate I Na kinetics with conformational changes within the 4 (DI-DIV) VSDs to define molecular mechanisms of Na V 1.5 modulation. Method and Results - Four Na V 1.5 constructs were created to track the voltage-dependent kinetics of conformational changes within each VSD, using voltage-clamp fluorometry. Each VSD displayed unique kinetics, consistent with distinct roles in determining I Na. In particular, DIII-VSD deactivation kinetics were modulated by depolarizing pulses with durations in the intermediate time domain that modulates late I Na. We then used the DII-VSD construct to probe the molecular pathology of 2 Brugada syndrome mutations (A735V and G752R). A735V shifted DII-VSD voltage dependence to depolarized potentials, whereas G752R significantly slowed DII-VSD kinetics. Both mutations slowed I Na activation, although 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 I Na activation and myocyte excitability. Conclusions - Our results reveal novel mechanisms whereby the Na V 1.5 VSDs regulate channel activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal Brugada syndrome mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications, and antiarrhythmic drugs alter Na V 1.5 at the molecular level.

Original languageEnglish
Pages (from-to)1228-1239
Number of pages12
JournalCirculation: Arrhythmia and Electrophysiology
Volume8
Issue number5
DOIs
StatePublished - Oct 1 2015

Keywords

  • Brugada syndrome
  • fluorometry
  • ion channels
  • mutation
  • sodium channels

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