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.

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...

Stim1 in the endoplasmic reticulum and the three Orai (also termed CRACM) channels in the plasma-membrane are main components of native Ca 2؉ release-activated Ca 2؉ channels. A pharmacological hallmark of these channels is their distinct sensitivity to 2-aminoethoxydiphenyl borate (2-APB). Here we report that Orai3 currents can be robustly stimulated by 75 M 2-APB independent of Stim1, whereas 2-APB at similar concentrations inhibited store-operated Orai1 currents. 2-APB did not only promote currents through Orai3 channels but also dramatically altered ion selectivity of Orai3 channels. This allowed for permeation of monovalent cations both in the inward as well as outward direction, which is in sharp contrast to the high Ca 2؉ selectivity of store-operated Orai3 currents. An Orai3-R66W mutant, which lacked in analogy to the severe combined immune deficiency mutant Orai1-R91W store-operated activation, was also found to be resistant to 2-APB stimulation. The change in selectivity by 2-APB was associated with an increase in Orai3 minimum pore size from about 3.8 Å to more than 5.34 Å . In line with a potential interaction of 2-APB with the Orai3 pore, among three pore mutants tested, the Orai3 E165Q mutant particularly resembled in its permeation properties those of 2-APB stimulated Orai3 and additionally exhibited a reduced response to 2-APB. In aggregate, stimulation of Orai3 currents by 2-APB occurred along with an alteration of the permeation pathway that represents a unique mechanism for regulating ion channel selectivity by chemical compounds.A major mechanism for many cell types to maintain longlasting elevation of intracellular Ca 2ϩ is the use of store-operated Ca 2ϩ influx (1). Initiated by the second messenger inositol 3-phosphate, Ca 2ϩ is released from the endoplasmic reticulum. Subsequently, Stim1 located in the endoplasmic reticulum senses via a luminal EF-hand the Ca 2ϩ content and redistributes after store-depletion to dynamically couple and activate the Orai1 channel, also named CRACM1 (2, 3). All three Orai members form highly Ca 2ϩ selective channels, and Orai1 has been suggested as the calcium release-activated calcium (CRAC) 6 channel in the plasma membrane (4, 5). Orai channels can be discriminated by distinct properties in feedback regulation to intracellular Ca 2ϩ and different pharmacological responses to 2-aminoethoxydiphenyl borate (2-APB; 50 M) (6, 7). Although Orai1 and Stim1 coexpressing cells were stimulated by low concentrations of 2-APB, high concentrations (50 M) completely inhibited Orai1 and partially suppressed Orai2 currents when coexpressed with Stim1 (6,8). This bimodal effect has also been observed for native CRAC channels in T-lymphocytes and mast cells (9). In contrast, coexpression of Orai1 and Stim2 resulted in robust stimulation by 2-APB (50 M), exhibiting a typical inward-rectifying current-voltage relationship of Orai1 (10, 11). It is proposed that 2-APB displaces the inhibitory calmodulin from the Stim2⅐Orai1 complex (10). Orai3, however, is exclusively stimula...

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca2؉ release-activated Ca 2؉ current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiledcoil domain C-terminally extended by ϳ50 amino acids and exhibits a strong binding to the Orai1 C terminus. Based on the homology within the Orai family, an analogous scenario might be assumed for Orai2 as well as Orai3 channels as both are activated in a similar STIM1-dependent manner. A combined approach of electrophysiology and Foerster resonance energy transfer microscopy uncovered a general mechanism in the communication of STIM1 with Orai proteins that involved the conserved putative coiled-coil domains in the respective Orai C terminus and the second coiled-coil motif in the STIM1 C terminus. A coiled-coil single mutation in the Orai1 C terminus abrogated communication with the STIM1 C terminus, whereas an analogous mutation in Orai2 and Orai3 still allowed for their moderate activation. However, increasing coiled-coil probability by a gain of function deletion in Orai1 or by generating an Orai1-Orai3 chimera containing the Orai3 C terminus recovered stimulation to a similar extent as with Orai2/3. At the level of STIM1, decreasing probability of the second coiled-coil domain by a single mutation within the STIM1 C terminus abolished activation of Orai1 but still enabled partial stimulation of Orai2/3 channels. A double mutation within the second coiled-coil motif of the STIM1 C terminus fully disrupted communication with all three Orai channels. In aggregate, the impairment in the overall communication between STIM1 and Orai channels upon decreasing probabilities of either one of the putative coiled-coil domains in the C termini might be compatible with the concept of their functional, heteromeric interaction.Store-operated Ca 2ϩ entry is a 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 4 years ago; the STIM1 (stromal interacting molecule 1) (7,8) and later on Orai1 (9 -11) have been identified as the two limiting components for CRAC activation. STIM1 is an ERlocated Ca 2ϩ sensor, and store depletion triggers its aggregation into punctae close to the plasma membrane, resulting in stimulation of CRAC currents (12,13). Its N terminus is located in the ER lumen and contains an EF-hand Ca 2ϩ -binding motif, which senses the ER Ca 2ϩ level, and a sterile ␣-motif, which is suggested to mediate homomeric STIM1 aggregation (14 -16). In the cytosolic STIM1 C terminus, two coiled-coil regions overlapping with the ezrin-radixin-moesin-like domain and a lysine-rich region are essential for CRAC activation (14,17,18). Three recent studies have independently identified the ezrinradixin-moesin ...

Patients with severe combined immune deficiency (SCID) suffer from defective T-cell Ca 2؉ signaling. A loss of Ca 2؉ entry has been linked at the molecular level to single missense mutation R91W in the store-operated Ca 2؉ channel ORAI1. However, the mechanistic impact of this mutation on ORAI1 function remains unclear. Confocal Förster resonance energy transfer microscopy revealed that dynamic store-operated coupling of STIM1 to ORAI1 R91W was largely sustained similar to wild-type ORAI1. Characterization of various point mutants at position 91 by whole cell patch clamp recordings displayed that neutral or even negatively charged amino acids did not abolish ORAI1 function. However, substitution by hydrophobic leucine, valine, or phenylalanine resulted in non-functional ORAI1 channels, despite preserved STIM1 coupling. Besides conformational constraints at the N terminus/membrane interface predicted for the hydrophobic mutants, additional key factor(s) were suggested to determine ORAI1 functionality. Calculation of the probability for the 1st transmembrane domain and its hydrophobicity revealed a substantial increase for all hydrophobic substitutions that lead to non-functional ORAI1 R91X mutants in contrast to those with hydrophilic residues. Hence, increased hydrophobicity might lead to disrupted permeation/gating, as an ORAI1 channel with increased pore size and R91W mutation failed to recover activity. In conclusion, the increase in hydrophobicity at the N terminus/ membrane interface represents the major cause for yielding nonfunctional ORAI1 channels.The immune system consists of various cell types such as Tand B-cells that are involved in protecting the body from foreign particles and pathogenic organisms. Defects in T-cell development impair normal immune function and may lead to primary immune deficiency. One subgroup thereof represented by the severe combined immunodeficiency (SCID) 4 occurs in 1 of 50,000 -100,000 live births, causing an onset of one or more serious infections, such as pneumonia, meningitis, or bloodstream infections, within the first few months of life (1, 2). It is currently known that defective T-cell signaling in SCID patients can arise from mutations in different genes including a point mutation in ORAI1 (3-5). T-cell function and proliferation requires calcium influx mediated by the Ca 2ϩ release-activated Ca 2ϩ channel. It is activated by depletion of intracellular Ca 2ϩ stores induced by the second messenger inositol 1,4,5-trisphosphate (6 -9) and this cytosolic Ca 2ϩ entry serves essential functions from secretion to gene expression and cell growth (10).A combination of RNA interference-based screening and analysis of single nucleotide polymorphism arrays of patients with SCID syndrome has led to the identification of the plasmamembrane protein ORAI1 as a key component of the Ca 2ϩ release-activated Ca 2ϩ channel complex (11,12). An overexpression of wild-type ORAI1 (4) or a related member ORAI3 (13) in SCID T-cells partially restored store-operated Ca 2ϩ influx. Based on p...