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

Sivakumar Namadurai, Dilshan Balasuriya, Rajit Rajappa, Martin Wiemhöfer, Katherine Stott, Jurgen Klingauf, J Michael Edwardson, Dimitri Y Chirgadze, Antony P Jackson

OAI: oai:www.repository.cam.ac.uk:1810/280466 • DOI: 10.17863/CAM.27837

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.

Analytical Ultracentrifugation Atomic force microscopy in vivo imaging Photoactivated Localization Microscopy Protein Trimerization Sodium Channels X-ray crystallography Amino Acid Sequence Binding Sites Cloning Molecular Crystallization Crystallography X-Ray Dimerization HEK293 Cells Humans Immunoglobulins Microscopy Atomic Force Molecular Sequence Data NAV1.5 Voltage-Gated Sodium Channel Protein Conformation Ultracentrifugation Voltage-Gated Sodium Channel beta-3 Subunit