Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly

• Published in: The Journal of biological chemistry 2014-04, Vol.289 (15), p.10797-10811
• Main Authors: Namadurai, Sivakumar, Balasuriya, Dilshan, Rajappa, Rajit, Wiemhöfer, Martin, Stott, Katherine, Klingauf, Jurgen, Edwardson, J.Michael, Chirgadze, Dimitri Y., Jackson, Antony P.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical Ultracentrifugation; Atomic Force Microscopy; Binding Sites; Cloning, Molecular; Crystallization; Crystallography, X-Ray; Dimerization; HEK293 Cells; Humans; Immunoglobulins - chemistry; In Vivo Imaging; Microscopy, Atomic Force; Molecular Sequence Data; NAV1.5 Voltage-Gated Sodium Channel - chemistry; Photoactivated Localization Microscopy; Protein Conformation; Protein Structure and Folding; Protein Trimerization; Sodium Channels; Ultracentrifugation; Voltage-Gated Sodium Channel beta-3 Subunit - chemistry; X-ray Crystallography
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.527994

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. Background: The vertebrate sodium channel β3 subunit regulates channel behavior. Results: The immunoglobulin domain of the human β3 subunit crystallizes as a trimer, and the full-length protein assembles as a trimer in vivo. Conclusion: Our results reveal an unexpected organization of the β3 subunit. Significance: A new structural insight into the sodium channel is presented.