Ca2+ entry through store-operated Ca 2+ release-activated Ca 2+ (CRAC) channels initiates key functions such as gene expression and exocytosis of inflammatory mediators. Activation of CRAC channels by store depletion involves the redistribution of the ER Ca 2+ sensor, stromal interaction molecule 1 (STIM1), to peripheral sites where it co-clusters with the CRAC channel subunit, Orai1. However, how STIM1 communicates with the CRAC channel and initiates the subsequent events culminating in channel opening is unclear. Here, we show that redistribution of STIM1 and Orai1 occurs in parallel with a pronounced increase in fluorescence resonance energy transfer (FRET) between STIM1 and Orai1, supporting the idea that activation of CRAC channels occurs through physical interactions with STIM1. Co-expression of Orai1-CFP and Orai1-YFP results in a high degree of FRET in resting cells, indicating that Orai1 exists as a multimer. However, store depletion triggers molecular rearrangements in Orai1 resulting in a decline in Orai1-Orai1 FRET. The decline in Orai1-Orai1 FRET is not seen in the absence of STIM1 co-expression and is abolished in Orai1 mutants with impaired STIM1 interaction. Both the STIM1-Orai1 interaction as well as the molecular rearrangements in Orai1 are altered by two powerful modulators of CRAC channel activity: extracellular Ca 2+ and 2-APB. These studies identify a STIM1-dependent conformational change in Orai1 during the activation of CRAC channels and reveal that STIM1-Orai1 interaction and the downstream Orai1 conformational change can be independently modulated to fine-tune CRAC channel activity.

Two defining functional features of ion channels are ion selectivity and channel gating. Ion selectivity is generally considered an immutable property of the open channel structure, whereas gating involves transitions between open and closed channel states typically without changes in ion selectivity 1. In store-operated Ca2+ release-activated Ca2+ (CRAC) channels, the molecular mechanism of channel gating by the CRAC channel activator, STIM1 (stromal interaction molecule 1) remains unknown. CRAC channels are distinguished by an extraordinarily high Ca2+ selectivity and are instrumental in generating sustained [Ca2+]i elevations necessary for gene expression and effector function in many eukaryotic cells 2. Here, we probed the central features of the STIM1 gating mechanism in the CRAC channel protein, Orai1, and identified V102, a residue located in the extracellular region of the pore, as a candidate for the channel gate. Mutations at V102 produced constitutively active CRAC channels that were open even in the absence of STIM1. Unexpectedly, although STIM1-free V102 mutant channels were not Ca2+-selective, their Ca2+ selectivity was dose-dependently boosted by interactions with STIM1. Similar enhancement of Ca2+ selectivity also occurred in wild-type (WT) Orai1 channels by increasing the number of STIM1 activation domains directly tethered to Orai1 channels. Thus, exquisite Ca2+ selectivity is not an intrinsic property of CRAC channels, but rather a tunable feature bestowed on otherwise non-selective Orai1 channels by STIM1. Our results demonstrate that STIM1-mediated gating of CRAC channels occurs through an unusual mechanism wherein permeation and gating are closely coupled.

Key points• 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.• 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.• 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.• These results help us understand the molecular requirements of STIM1-mediated activation of Orai1 channels and regulation of channel ion selectivity.Abstract Ca 2+ release-activated Ca 2+ (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) Ca 2+ 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 (73)(74)(75)(76)(77)(78)(79)(80)(81)(82)(83)(84)(85)(272)(273)(274)(275)(276)(277)(278)(279), 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 B. A. McNally, A. Somasundaram and A. Jairaman contributed equally to this work.

Calcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca 2ϩ signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca

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