Store-operated Ca 2+ entry is mediated by Ca 2+ release-activated Ca 2+ (CRAC) channels following Ca 2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca 2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.Receptor-mediated signaling in nonexcitable cells, immune cells in particular, involves an initial rise in intracellular Ca 2+ due to release from the intracellular stores. The resulting depletion of the intracellular stores induces Ca 2+ entry through the plasma membrane through CRAC channels (1-4). This phenomenon is central to many physiological processes such as T cell proliferation, gene transcription, and cytokine release (3, 5-7). Biophysically, CRAC currents have been well characterized (2,8,9), but the identity of the CRAC channel itself and the pathway resulting in its activation are still unknown. Recently, STIM1 (for stromal interaction molecule in Drosophila) was identified as an essential component of store-operated calcium entry (10,11). This protein is located in intracellular compartments that likely represent parts of the endoplasmic reticulum (ER). It has a single transmembranespanning domain with a C-terminal Ca 2+ -binding motif that appears to be crucial for its hypothesized function as the ER sensor for luminal Ca 2+ concentration. When stores become depleted, STIM1 redistributes into distinct structures (punctae) that move toward Fig. 1, B and C, from cells treated with dsRNA against Rho1 (mock) and stim1, as well as two genes we later identified as CRAC modulators 1 and 2 (CRACM1 and CRACM2). On the basis of inhibitory efficacy relative to positive and negative controls, we identified ~1500 genes that reduced Ca 2+ influx to varying degrees (table S1). After eliminating numerous genes based on artifactual fluorescence signals or because they represent known housekeeping genes, cell cycle regulators, and so on, we eventually arrived at 27 candidate genes (table S2) that were subsequently evaluated in a secondary screen using single-cell patch-clamp assays.From the secondary patch-clamp screen, we identified two novel genes that are essential for CRAC channel function, CRACM1 (encoded by olf186-F in Drosophila and FLJ14466 in human) and CRACM2 (encoded by dpr3 in Drosophila, with no human ortholog). We measured CRAC currents in Drosophila Kc cells after inositol 1,4,5-trisphosphate (IP 3 )-mediated depletion of Ca 2+ from intracellular stores. Both untreated control wild-type cells and cells treated with an irrelevant dsRNA against Rho1 (mock) responded by ra...

Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I CRAC ) [1][2][3][4] . However, individual overexpression of either protein fails to significantly amplify I CRAC . Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I CRAC , suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.Receptor-mediated release of Ca 2+ from intracellular stores induces Ca 2+ entry through calcium release-activated calcium (CRAC) channels [5][6][7] . Previous studies have identified STIM1 as the potential sensor for endoplasmic reticulum luminal Ca 2+ concentration 1,8,9 . When Ca 2+ is depleted from intracellular stores, STIM1 translocates to vesicular structures (punctae) underneath the plasma membrane, where it is hypothesized to activate CRAC channels residing in the plasma membrane. A second protein, CRACM1, has recently been identified as essential for activating CRAC channels 3,4 . This protein contains four transmembrane domains, is located in the plasma membrane and, therefore, may represent the CRAC channel itself, a subunit of the channel, or a regulatory molecule that couples to the channel. When overexpressed individually, neither STIM1 nor CRACM1 can significantly potentiate I CRAC 1-4 .To address the potential interaction of STIM1 and CRACM1, both proteins were overexpressed individually, or in combination, in HEK293 and Jurkat T cells and the CRAC (Fig. 1b, d). Consistent with previous work 1,9 , overexpression of STIM1 alone caused a small-to-modest increase in I CRAC in HEK293 and Jurkat cells (Fig. 1a, c). CRACM1 overexpression alone did not affect the CRAC currents induced by store depletion in HEK293 cells (Fig. 1a, b) and caused a small reduction in I CRAC in Jurkat cells (Fig. 1c, d). Unless simply due to a general effect of transfection or variability of I CRAC across preparations, this reduction may be due to some kind of dominant-negative effect. Taken together, the available data on CRACM1 and STIM1 suggest that the individually expressed proteins, although essential for I CRAC manifestation, cannot significantly amplify the current. This would indicate that these proteins are either not sufficient to generate large CRAC currents or that they are stoichiometrically linked and limit each others' ability to generate CRAC currents above normal. Therefore, we co-overexpressed both proteins in HEK293 cells (see Supplementary Information, Fig. S1) and assessed store-operated currents by patch clamp. HHS Public AccessThe co-overexpression of STIM1 and CRACM1, in both HEK293 and Jurkat cells, is sufficient to generate enormous membrane currents ...

Cutaneous mast cell responses to physical (thermal, mechanical, or osmotic) stimuli underlie the pathology of physical urticarias. In vitro experiments suggest that mast cells respond directly to these stimuli, implying that a signaling mechanism couples functional responses to physical inputs in mast cells. We asked whether transient receptor potential (vanilloid) (TRPV) cation channels were present and functionally coupled to signaling pathways in mast cells, since expression of this channel subfamily confers sensitivity to thermal, osmotic, and pressure inputs. Transcripts for a range of TRPVs were detected in mast cells, and we report the expression, surface localization, and oligomerization of TRPV2 protein subunits in these cells. We describe the functional coupling of TRPV2 protein to calcium fluxes and proinflammatory degranulation events in mast cells. In addition, we describe a novel protein kinase A (PKA)–dependent signaling module, containing PKA and a putative A kinase adapter protein, Acyl CoA binding domain protein (ACBD)3, that interacts with TRPV2 in mast cells. We propose that regulated phosphorylation by PKA may be a common pathway for TRPV modulation.

Cannabinoid modulation of immune responses is a pathological consequence of marijuana abuse and a potential outcome of therapeutic application of the drug. Moreover, endogenous cannabinoids are physiological immune regulators. In the present report, we describe alterations in gene transcription that occur after cannabinoid exposure in a mast cell line, RBL2H3. Cannabinoid exposure causes marked changes in the transcript levels for numerous genes, acting both independently of and in concert with immunoreceptor stimulation via FcεRI. In two mast cell lines, we observed mRNA and protein expression corresponding to both CB1 and CB2 cannabinoid receptor isoforms, contrary to the prevailing view that CB1 is restricted to the CNS. We show that coexpression of the two isoforms is not functionally redundant in mast cells. Analysis of signaling pathways downstream of cannabinoid application reveals that activation of extracellular signal-regulated kinase, AKT, and a selected subset of AKT targets is accomplished by CB2 ligands and nonselective CB1/CB2 agonists in mast cells. CB1 inhibition does not affect AKT or extracellular signal-regulated kinase activation by cannabinoids, indicating that CB2 is the predominant regulatory receptor for these kinases in this cell context. CB1 receptors are, however, functional in these mast cells, since they can contribute to suppression of secretory responses.

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The transient receptor potential, sub-family Vanilloid (TRPV)(2) cation channel is activated in response to extreme temperature elevations in sensory neurons. However, TRPV2 is widely expressed in tissues with no sensory function, including cells of the immune system. Regulation of GRC, the murine homolog of TRPV2 has been studied in insulinoma cells and myocytes. GRC is activated in response to certain growth factors and neuropeptides, via a mechanism that involves regulated access of the channel to the plasma membrane. This is likely to be an important primary control mechanism for TRPV2 outside the CNS. Here, we report that a regulated trafficking step controls the access of TRPV2 to the cell surface in mast cells. In mast cells, elevations in cytosolic cAMP are sufficient to drive plasma membrane localization of TRPV2. We have previously proposed that the recombinase gene activator protein (RGA), a four-transmembrane domain, intracellular protein, associates with TRPV2 during the biosynthesis and early trafficking of the channel. We use a polyclonal antibody to RGA to confirm the formation of a physiological complex between RGA and TRPV2. Finally, we show that over-expression of the RGA protein potentiates the basal surface localization of TRPV2. We propose that trafficking and activation mechanisms intersect for TRPV2, and that cAMP mobilizing stimuli may regulate TRPV2 localization in non-sensory cells. RGA participates in the control of TRPV2 surface levels, and co-expression of RGA may be a key component of experimental systems that seek to study TRPV2 physiology.

TRPV ion channels transduce a range of temperature stimuli. We proposed that analysis of the protein-protein interactions made by TRPV2 might give insight into the key issues surrounding this channel. These issues include the potential functional significance of TRPV2 in non-sensory tissues, the molecules involved in transducing its activation signal(s) and the mechanism by which its trafficking to the cell surface is regulated. Here we describe the interaction of TRPV2 channel with the RGA gene product. RGA is a four-transmembrane domain, intracellularly localized protein. RGA associates with TRPV2 in a rat mast cell line that is a native context for both proteins. The interaction between TRPV2 and RGA is transient and occurs intracellularly. RGA does not accompany TRPV2 to the cell surface. Formation of the TRPV2/RGA complex is dependent upon a cellular glycosylation event, suggesting that RGA may play a chaperone or targeting role for TRPV2 during the maturation of the ion channel protein. These data record a novel protein-protein interaction for TRPV2 and provide a foundation for future study of the potential regulatory contribution of RGA to TRPV2 function.