Crystal structure and molecular imaging of the Nav channel β3 subunit indicates a trimeric assembly

J Biol Chem. 2014 Apr 11;289(15):10797-10811. doi: 10.1074/jbc.M113.527994. Epub 2014 Feb 24.

Abstract

The vertebrate sodium (Nav) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Nav 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations.

Keywords: Analytical Ultracentrifugation; Atomic Force Microscopy; In Vivo Imaging; Photoactivated Localization Microscopy; Protein Trimerization; Sodium Channels; X-ray Crystallography.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Cloning, Molecular
  • Crystallization
  • Crystallography, X-Ray
  • Dimerization
  • HEK293 Cells
  • Humans
  • Immunoglobulins / chemistry
  • Microscopy, Atomic Force
  • Molecular Sequence Data
  • NAV1.5 Voltage-Gated Sodium Channel / chemistry
  • Protein Conformation
  • Ultracentrifugation
  • Voltage-Gated Sodium Channel beta-3 Subunit / chemistry*

Substances

  • Immunoglobulins
  • NAV1.5 Voltage-Gated Sodium Channel
  • SCN5A protein, human
  • Voltage-Gated Sodium Channel beta-3 Subunit

Associated data

  • PDB/4L1D