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 Ca(2+) 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.

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Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.

Background and purpose: Pyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill‐defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation.Experimental approach: Pyrazoles were generated by microwave‐assisted synthesis and tested for effects on Ca²⁺ entry by Fura‐2 imaging and membrane currents by patch‐clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL‐2H3 mast cells, which express classical store‐operated Ca²⁺ entry mediated by Orai channels. The consequences of inhibitory effects on Ca²⁺ signalling in RBL‐2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T‐cells activation.Key Results: Pyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1‐ and TRPC3‐mediated Ca²⁺ entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37‐fold higher potency to inhibit Orai1‐mediated Ca²⁺ entry as compared with TRPC3‐mediated Ca²⁺ entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3‐mediated responses (18‐fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation.Conclusions and Implications: The pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai‐mediated Ca²⁺ entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca²⁺ channels.

STIM1 and Orai1 have been reported to interact upon store depletion culminating in Ca(2+) release-activated Ca(2+) current activation. Recently, the essential region has been identified within the STIM1 C terminus that includes the second coiled-coil domain C-terminally extended by approximately 50 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.