Direct Measurement of Cardiac Na+ Channel Conformations Reveals Molecular Pathologies of Inherited Mutations.
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 4 (DI-DIV) VSDs to define molecular mechanisms of NaV1.5 modulation.


RESULTS
Four NaV1.5 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 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 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 INa 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 INa activation and myocyte excitability.


CONCLUSIONS
Our results reveal novel mechanisms whereby the NaV1.5 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 NaV1.5 at the molecular level.
From the Department of Biomedical Engineering, Washington University in St. Louis, MO (Z.V., W.Z., A.R.S., J.L.P., A.K., E.J.H., M.A.Z., J.C., J.R.S.); and MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary (Z.V.).
Zoltan
Z
Varga
From the Department of Biomedical Engineering, Washington University in St. Louis, MO (Z.V., W.Z., A.R.S., J.L.P., A.K., E.J.H., M.A.Z., J.C., J.R.S.); and MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary (Z.V.).
Wandi
W
Zhu
From the Department of Biomedical Engineering, Washington University in St. Louis, MO (Z.V., W.Z., A.R.S., J.L.P., A.K., E.J.H., M.A.Z., J.C., J.R.S.); and MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary (Z.V.).
Angela R
AR
Schubert
From the Department of Biomedical Engineering, Washington University in St. Louis, MO (Z.V., W.Z., A.R.S., J.L.P., A.K., E.J.H., M.A.Z., J.C., J.R.S.); and MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary (Z.V.).
Jennifer L
JL
Pardieck
From the Department of Biomedical Engineering, Washington University in St. Louis, MO (Z.V., W.Z., A.R.S., J.L.P., A.K., E.J.H., M.A.Z., J.C., J.R.S.); and MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary (Z.V.).
Arie
A
Krumholz
From the Department of Biomedical Engineering, Washington University in St. Louis, MO (Z.V., W.Z., A.R.S., J.L.P., A.K., E.J.H., M.A.Z., J.C., J.R.S.); and MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary (Z.V.).
Eric J
EJ
Hsu
From the Department of Biomedical Engineering, Washington University in St. Louis, MO (Z.V., W.Z., A.R.S., J.L.P., A.K., E.J.H., M.A.Z., J.C., J.R.S.); and MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary (Z.V.).
Mark A
MA
Zaydman
From the Department of Biomedical Engineering, Washington University in St. Louis, MO (Z.V., W.Z., A.R.S., J.L.P., A.K., E.J.H., M.A.Z., J.C., J.R.S.); and MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary (Z.V.).
Jianmin
J
Cui
From the Department of Biomedical Engineering, Washington University in St. Louis, MO (Z.V., W.Z., A.R.S., J.L.P., A.K., E.J.H., M.A.Z., J.C., J.R.S.); and MTA-DE-NAP B Ion Channel Structure-Function Research Group, RCMM, Department of Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary (Z.V.). jonsilva@wustl.edu.
Jonathan R
JR
Silva
D053840:Brugada Syndrome / Q000235:genetics* / Q000503:physiopathology; D020022:Genetic Predisposition to Disease; D006801:Humans; D015640:Ion Channel Gating; D007700:Kinetics; D008564:Membrane Potentials / Q000235:genetics / Q000502:physiology; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D015222:Sodium Channels / Q000235:genetics* / Q000502:physiology
D015222:Sodium Channels
Brugada syndrome; fluorometry; ion channels; mutation; sodium channels