STIM1 and ORAI1 (also termed CRACM1) are essential components of the classical calcium release-activated calcium current; however, the mechanism of the transmission of information of STIM1 to the calcium release-activated calcium/ORAI1 channel is as yet unknown. Here we demonstrate by Förster resonance energy transfer microscopy a dynamic coupling of STIM1 and ORAI1 that culminates in the activation of Ca 2؉ entry. Förster resonance energy transfer imaging of living cells provided insight into the time dependence of crucial events of this signaling pathway comprising Ca 2؉ store depletion, STIM1 multimerization, and STIM1-ORAI1 interaction. Accelerated store depletion allowed resolving a significant time lag between STIM1-STIM1 and STIM1-ORAI1 interactions. Store refilling reversed both STIM1 multimerization and STIM1-ORAI1 interaction. The cytosolic STIM1 C terminus itself was able, in vitro as well as in vivo, to associate with ORAI1 and to stimulate channel function, yet without ORAI1-STIM1 cluster formation. The dynamic interaction occurred via the C terminus of ORAI1 that includes a putative coiled-coil domain structure. An ORAI1 C terminus deletion mutant as well as a mutant (L273S) with impeded coiled-coil domain formation lacked both interaction as well as functional communication with STIM1 and failed to generate Ca 2؉ inward currents. An N-terminal deletion mutant of ORAI1 as well as the ORAI1 R91W mutant linked to severe combined immune deficiency syndrome was similarly impaired in terms of current activation despite being able to interact with STIM1. Hence, the C-terminal coiled-coil motif of ORAI1 represents a key domain for dynamic coupling to STIM1.

STIM1 couples to ORAI1 via an intramolecular transition into an extended conformationUpon depletion of ER calcium stores, STIM1 and ORAI1 associate and induce calcium release-activated calcium (CRAC) currents. This study reveals that STIM1 undergoes an intramolecular transition into an extended conformation that is involved in ORAI1 binding and activation.

In immune cells, generation of sustained Ca2؉ levels is mediated by the Ca 2؉ release-activated Ca 2؉ (CRAC) current. Molecular key players in this process comprise the stromal interaction molecule 1 (STIM1) that functions as a Ca 2؉ sensor in the endoplasmic reticulum and ORAI1 located in the plasma membrane. Depletion of endoplasmic reticulum Ca 2؉ stores leads to STIM1 multimerization into discrete puncta, which co-cluster with ORAI1 to couple to and activate ORAI1 channels. The cytosolic C terminus of STIM1 is sufficient to activate ORAI1 currents independent of store depletion. Here we identified an ORAI1-activating small fragment (OASF, amino acids 233-450/474) within STIM1 C terminus comprising the two coiled-coil domains and additional 50 -74 amino acids that exhibited enhanced interaction with ORAI1, resulting in 3-fold increased Ca 2؉ currents. This OASF, similar to the complete STIM1 C terminus, displayed the ability to homomerize by a novel assembly domain that occurred subsequent to the coiled-coil domains. A smaller fragment (amino acids 233-420) generated by a further deletion of 30 amino acids substantially reduced the ability to homomerize concomitant to a loss of coupling to as well as activation of ORAI1. Extending OASF by 35 amino acids (233-485) did not alter homomerization but substantially decreased efficiency in coupling to and activation of ORAI1. Expressing OASF in rat basophilic leukemia (RBL) mast cells demonstrated its enhanced plasma membrane targeting associated with 2.5-fold larger CRAC currents in comparison with the complete STIM1 C terminus. In aggregate, we have identified two cytosolic key regions within STIM1 C terminus that control ORAI1/CRAC activation: a homomerization domain indispensable for coupling to ORAI1 and a modulatory domain that controls the extent of coupling to ORAI1.Store-operated Ca 2ϩ entry is key to cellular regulation of short term responses such as contraction and secretion as well as long term processes like proliferation and cell growth (1). The prototypic and best characterized store-operated channel is the Ca 2ϩ release-activated Ca 2ϩ (CRAC) 5 channel (2-6). However, its molecular components have remained elusive until 3 years ago; the stromal interacting molecule 1 (STIM1) (7, 8) and later on ORAI1 (9 -11) have been identified as the two limiting components for CRAC activation. STIM1 is an ER-located Ca 2ϩ sensor (7,8,12), and store depletion triggers its aggregation into puncta close to the plasma membrane, resulting in stimulation of CRAC currents (13,14). Its N terminus is located in the ER lumen and contains an EF-hand Ca 2ϩ binding motif that senses the ER Ca 2ϩ level and a sterile ␣ motif that is suggested to mediate homomeric STIM1 aggregation (15, 16). In the cytosolic STIM1 C terminus, two coiled-coil regions overlapping with the ezrin-radixin-moesin (ERM)-like domain and a lysine-rich region have been proposed as essential for CRAC activation (15,17,18). ORAI1 has been assumed to act in concert with STIM1 (10,19,20), activating inward Ca...

Background: STIM1 and Orai1, reconstituting a main cellular Ca2+ entry pathway, interact via their cytosolic strands.Results: The extended transmembrane Orai1 N-terminal (ETON) region combines binding interface and gate for Orai1 activation by STIM1.Conclusion: Several “hot spot” residues in the ETON region mediate STIM1 interaction, enabling conformational reorientation of the gate.Significance: Identification of critical residues for protein-protein interaction are fundamental to therapeutic drug development.

The channel Orai1 requires Ca store depletion in the endoplasmic reticulum and an interaction with the Ca sensor STIM1 to mediate Ca signaling. Alterations in Orai1-mediated Ca influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca permeation associated with gene transcription and provide a gating mechanism for Orai1.