Crystal Structure and Molecular Imaging of the Na _v Channel β3 Subunit Indicates a Trimeric Assembly*

Abstract

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.

Abstract

The vertebrate sodium (Na _v) 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 Na _v 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 Na _v 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 Na _v channel cross-linking and provide new structural insights into some pathological Na _v channel mutations.