Store-operated calcium entry (SOCE) is a ubiquitous mechanism that is mediated by distinct SOC channels, ranging from the highly selective calcium release-activated Ca²⁺ (CRAC) channel in rat basophilic leukemia and other hematopoietic cells to relatively Ca²⁺-selective or non-selective SOC channels in other cells. Although the exact composition of these channels is not yet established, TRPC1 contributes to SOC channels and regulation of physiological function of a variety of cell types. Recently, Orai1 and STIM1 have been suggested to be sufficient for generating CRAC channels. Here we show that Orai1 and STIM1 are also required for TRPC1-SOC channels. Knockdown of TRPC1, Orai1, or STIM1 attenuated, whereas overexpression of TRPC1, but not Orai1 or STIM1, induced an increase in SOC entry and I_SOC in human salivary gland cells. All three proteins were co-localized in the plasma membrane region of cells, and thapsigargin increased co-immunoprecipitation of TRPC1 with STIM1, and Orai1 in human salivary gland cells as well as dispersed mouse submandibular gland cells. In aggregate, the data presented here reveal that all three proteins are essential for generation of I_SOC in these cells and that dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in activation of SOC channel in response to internal Ca²⁺ store depletion. Thus, these data suggest a common molecular basis for SOC and CRAC channels.

Store-operated Ca²⁺ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca²⁺ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I_SOC, activated in response to Ca²⁺ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I_CRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(⁶⁸⁴EE⁶⁸⁵). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca²⁺ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd³⁺, removal of extracellular Ca²⁺, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca²⁺-containing, but not Ca²⁺-free, medium. Consistent with this, I_CRAC is activated in cells pretreated with thapsigargin in Ca²⁺-free medium while I_SOC is activated in cells pretreated in Ca²⁺-containing medium. Significantly, TRPC1 function is required for sustained K_Ca activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca²⁺ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca²⁺ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE. Store-operated Ca²⁺ entry is present in all cell types and determines sustained cytosolic [Ca²⁺] increases that are critical for regulating a wide variety of physiological functions. This Ca²⁺ entry mechanism is activated in response to depletion of Ca²⁺ in the endoplasmic reticulum (ER). When ER [Ca²⁺] is decreased, the Ca²⁺-sensor protein STIM1 aggregates in the ER membrane and moves to regions in the periphery of the cells where it interacts with and activates two major types of channels that contribute to store-operated Ca²⁺ entry: CRAC and SOC. While gating of Orai1 by STIM1 is sufficient for CRAC channel activity, both Orai1 and transient receptor potential channel 1 (TRPC1) contribute to SOC channel function. The molecular composition of SOC channels and the critical role of Orai1 in activation of TRPC1 have not yet been established. In this study, we demonstrate that TRPC1 and Orai1 are components of distinct channels, both of which are regulated by STIM1. Importantly, we show that Orai1-mediated Ca²⁺ entry triggers plasma membrane insertion of TRPC1 which is then gated by STIM1. Ca²⁺ entry via functional TRPC1-STIM1 channels provides additional increase in cytosolic [Ca²⁺] that is required for regulation of specific cell functions such as K_Ca activation. Together, our findings elucidate the critical role of Orai1 in TRPC1 channel function. We suggest that the regulation of TRPC1 trafficking provides a mechanism for rapidly modulating cytosolic [Ca²⁺] following Ca²⁺ store depletion.

Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca²⁺ sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca²⁺ entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca²⁺ entry and activated typical I_CRAC current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, I_SOC, that was blocked by 1 μm Gd³⁺ and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and I_SOC in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and I_SOC, whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE.

In vivo recycling of nitrate (NO ₃ ⁻ ) and nitrite (NO ₂ ⁻ ) is an important alternative pathway for the generation of nitric oxide (NO) and maintenance of systemic nitrate–nitrite–NO balance. More than 25% of the circulating NO ₃ ⁻ is actively removed and secreted by salivary glands. Oral commensal bacteria convert salivary NO ₃ ⁻ to NO ₂ ⁻ , which enters circulation and leads to NO generation. The transporters for NO ₃ ⁻ in salivary glands have not yet been identified. Here we report that sialin ( SLC17A 5 ), mutations in which cause Salla disease and infantile sialic acid storage disorder (ISSD), functions as an electrogenic 2NO ₃ ⁻ /H ⁺ cotransporter in the plasma membrane of salivary gland acinar cells. We have identified an extracellular pH-dependent anion current that is carried by NO ₃ ⁻ or sialic acid (SA), but not by Br ⁻ , and is accompanied by intracellular acidification. Both responses were reduced by knockdown of sialin expression and increased by the plasma membrane-targeted sialin mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed reduced SA- and NO ₃ ⁻ -induced currents compared with healthy controls. Furthermore, expression of disease-associated sialin mutants in fibroblasts and salivary gland cells suppressed the H ⁺ -dependent NO ₃ ⁻ conductance. Importantly, adenovirus-dependent expression of the sialinH183R mutant in vivo in pig salivary glands decreased NO ₃ ⁻ secretion in saliva after intake of a NO ₃ ⁻ -rich diet. Taken together, these data demonstrate that sialin mediates nitrate influx into salivary gland and other cell types. We suggest that the 2NO ₃ ⁻ /H ⁺ transport function of sialin in salivary glands can contribute significantly to clearance of serum nitrate, as well as nitrate recycling and physiological nitrite-NO homeostasis.