4.6 Article

Gating control of the cardiac sodium channel Nav1.5 by its?3-subunit involves distinct roles for a transmembrane glutamic acid and the extracellular domain

Journal

JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 294, Issue 51, Pages 19752-19763

Publisher

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA119.010283

Keywords

sodium channel; electrophysiology; protein structure; fluorescence; cardiomyopathy; cardiovascular disease; voltage clamp fluorescence

Funding

  1. Medical Research Council [MR/M001288/1]
  2. Wellcome Trust [105727/Z/14/Z]
  3. British Heart Foundation [PG/14/79/31102]
  4. American Heart Association [15PRE-25080073]
  5. National Institutes of Health [R01 HL136553]
  6. Islamic Development Bank
  7. Cambridge International Trust
  8. Department of Biochemistry, University of Cambridge
  9. MRC [MR/M001288/1] Funding Source: UKRI

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The auxiliary ?3-subunit is an important functional regulator of the cardiac sodium channel Nav1.5, and some ?3 mutations predispose individuals to cardiac arrhythmias. The ?3-subunit uses its transmembrane ?-helix and extracellular domain to bind to Nav1.5. Here, we investigated the role of an unusually located and highly conserved glutamic acid (Glu-176) within the ?3 transmembrane region and its potential for functionally synergizing with the ?3 extracellular domain (ECD). We substituted Glu-176 with lysine (E176K) in the WT ?3-subunit and in a ?3-subunit lacking the ECD. Patch-clamp experiments indicated that the E176K substitution does not affect the previously observed ?3-dependent depolarizing shift of V-? of steady-state inactivation but does attenuate the accelerated recovery from inactivation conferred by the WT ?3-subunit. Removal of the ?3-ECD abrogated both the depolarizing shift of steady-state inactivation and the accelerated recovery, irrespective of the presence or absence of the Glu-176 residue. We found that steady-state inactivation and recovery from inactivation involve movements of the S4 helices within the DIII and DIV voltage sensors in response to membrane potential changes. Voltage-clamp fluorometry revealed that the E176K substitution alters DIII voltage sensor dynamics without affecting DIV. In contrast, removal of the ECD significantly altered the dynamics of both DIII and DIV. These results imply distinct roles for the ?3-Glu-176 residue and the ?3-ECD in regulating the conformational changes of the voltage sensors that determine channel inactivation and recovery from inactivation.

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