title: Crystal structure and molecular imaging of the Nav channel β3 subunit indicates a trimeric assembly.
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.
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom.
authors.forename: Sivakumar
authors.initials: S
authors.lastname: Namadurai
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
authors.forename: Dilshan
authors.initials: D
authors.lastname: Balasuriya
authors.affiliation: Institute of Medical Physics and Biophysics, University of Münster, Robert-Koch Strasse, 31 48149 Münster, Germany.
authors.forename: Rajit
authors.initials: R
authors.lastname: Rajappa
authors.affiliation: Institute of Medical Physics and Biophysics, University of Münster, Robert-Koch Strasse, 31 48149 Münster, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Wiemhöfer
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom.
authors.forename: Katherine
authors.initials: K
authors.lastname: Stott
authors.affiliation: Institute of Medical Physics and Biophysics, University of Münster, Robert-Koch Strasse, 31 48149 Münster, Germany.
authors.forename: Jurgen
authors.initials: J
authors.lastname: Klingauf
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
authors.forename: J Michael
authors.initials: JM
authors.lastname: Edwardson
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom. Electronic address: dyc21@cam.ac.uk.
authors.forename: Dimitri Y
authors.initials: DY
authors.lastname: Chirgadze
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom. Electronic address: apj10@cam.ac.uk.
authors.forename: Antony P
authors.initials: AP
authors.lastname: Jackson
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D019281:Dimerization; D057809:HEK293 Cells; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000737:chemistry; D011487:Protein Conformation; D014461:Ultracentrifugation; D062549:Voltage-Gated Sodium Channel beta-3 Subunit / Q000737:chemistry*
chemical_list: D007136:Immunoglobulins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; D062549:Voltage-Gated Sodium Channel beta-3 Subunit
keywords: Analytical Ultracentrifugation; Atomic Force Microscopy; In Vivo Imaging; Photoactivated Localization Microscopy; Protein Trimerization; Sodium Channels; X-ray Crystallography