The C- and N-terminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels

Key points center dot The endoplasmic reticulum protein, stromal interaction molecule 1 (STIM1), activates Orai1 channels by directly interacting with each Orai1 subunit at the C- and N-termini. Current models about the roles of these sites are rooted in notions of modularity, with the C-terminal site thought to mediate STIM1 binding and the N-terminal site thought to regulate channel gating. center dot Here we report that the functions of the two sites are not so distinct: the N-terminal site contributes to the stable association of STIM1 to Orai1, and, conversely, the C-terminal site regulates channel activation. center dot In addition to channel activation, STIM1 binding also modulates Orai1 channel ion selectivity. The structural requirements for modulation of ion selectivity closely match those seen for gating, suggesting that gating and permeation are closely coupled in Orai1 channels. center dot These results help us understand the molecular requirements of STIM1-mediated activation of Orai1 channels and regulation of channel ion selectivity. Abstract Ca2+ release-activated Ca2+ (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca2+ sensor, and Orai1, the CRAC channel protein, at overlapping sites in the ER and plasma membranes (PMs). The redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, an important event that not only controls gating, but also regulates Orai1 ion selectivity. Orai1 harbours two STIM1 binding sites, one each on the intracellular C- and N-termini. Previous studies have proposed modular functions for these sites, with the C-terminal site thought to regulate STIM1-Orai1 binding and trapping of Orai1 at the ER-PM junctions, and the N-terminal site mediating gating. However, here we find that a variety of mutations in the N-terminal site impair the binding of Orai1 to STIM1 and to the soluble CRAC activation domain (CAD). Gating could be restored in several N- and C-terminal point mutants by directly tethering the minimal STIM1 activation domain (S) to Orai1 (Orai1-SS channels), indicating that loss of gating in these mutants by full-length STIM1 results from insufficient ligand binding. By contrast, gating could not be restored in mutant Orai1-SS channels carrying more drastic deletions that removed the STIM1 binding sites (1-85, 73-85, or 272-279 Orai1), suggesting that STIM1 binding to both sites is essential for channel activation. Moreover, analysis of ion selectivity indicated that the molecular requirements for gating and modulation of ion selectivity are similar, yet substantively different from those for Orai1 puncta formation, suggesting that ion selectivity and gating are mechanistically coupled in CRAC channels. Our results indicate that the C- and N-terminal STIM1 binding sites are both essential for multiple aspects of Orai1 function including STIM1-Orai1 association, Orai1 trapping, and channel activation.