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

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

Atomic force microscopy (AFM) imaging suggests that stromal interaction molecule 1 (STIM1) binds to Orai1 with sixfold symmetry

Depletion of Ca(2+) from the endoplasmic reticulum (ER) lumen triggers the opening of Ca(2+) release-activated Ca(2+) (CRAC) channels at the plasma membrane. CRAC channels are activated by stromal interaction molecule 1 (STIM1), an ER resident protein that senses Ca(2+) store depletion and interacts with Orai1, the pore-forming subunit of the channel. The subunit stoichiometry of the CRAC channel is controversial. Here we provide evidence, using atomic force microscopy (AFM) imaging, that Orai1 assembles as a hexamer, and that STIM1 binds to Orai1 with sixfold symmetry. STIM1 associates with Orai1 in the form of monomers, dimers, and multimeric string-like structures that form links between the Orai1 hexamers. Our results provide new insights into the nature of the interactions between STIM1 and Orai1

Sigma1 receptors inhibit store-operated Ca2+ entry by attenuating coupling of STIM1 to Orai1.

Sigma1 receptors (σ1Rs) are expressed widely; they bind diverse ligands, including psychotropic drugs and steroids, regulate many ion channels, and are implicated in cancer and addiction. It is not known how σ1Rs exert such varied effects. We demonstrate that σ1Rs inhibit store-operated Ca(2+)entry (SOCE), a major Ca(2+)influx pathway, and reduce the Ca(2+)content of the intracellular stores. SOCE was inhibited by expression of σ1R or an agonist of σ1R and enhanced by loss of σ1R or an antagonist. Within the endoplasmic reticulum (ER), σ1R associated with STIM1, the ER Ca(2+)sensor that regulates SOCE. This interaction was modulated by σ1R ligands. After depletion of Ca(2+)stores, σ1R accompanied STIM1 to ER-plasma membrane (PM) junctions where STIM1 stimulated opening of the Ca(2+)channel, Orai1. The association of STIM1 with σ1R slowed the recruitment of STIM1 to ER-PM junctions and reduced binding of STIM1 to PM Orai1. We conclude that σ1R attenuates STIM1 coupling to Orai1 and thereby inhibits SOCE.We thank T.P. Su (National Institutes of Health, USA) for σ1R siRNA and control plasmids, S. Srikanth and Y. Gwack (University of Los Angeles California, USA) for the Orai1E106Q 28 construct, A. Parekh (University of Oxford, UK) for the GFP-NFAT plasmid, P. Hogan (La Jolla Institute for Allergy and Immunology, USA) for the CAD construct and D. M. F. Cooper (University of Cambridge, UK) for the Orai1-CFP construct. S.S. is supported by the Cambridge International and European Trust, D.B. by a David James Bursary from the Department of Pharmacology, Cambridge, and G.V. by the Jardines Matheson student bursary. This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/J018236/1 to J.M.E and BB/F001320/1 to R.D.M.L.), and the Wellcome Trust (101844 to C.W.T). The authors declare no competing financial interests.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Rockefeller University Press