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...

contributed equally to this work TIA-1 and TIAR are related proteins that bind to an AU-rich element (ARE) in the 3¢ untranslated region of tumor necrosis factor alpha (TNF-a) transcripts. To determine the functional signi®cance of this interaction, we used homologous recombination to produce mutant mice lacking TIA-1. Although lipopolysaccharide (LPS)-stimulated macrophages derived from wild-type and TIA-1 ±/± mice express similar amounts of TNF-a transcripts, macrophages lacking TIA-1 produce signi®cantly more TNF-a protein than wild-type controls. The half-life of TNF-a transcripts is similar in wild-type and TIA-1 ±/± macrophages, indicating that TIA-1 does not regulate transcript stability. Rather, the absence of TIA-1 signi®cantly increases the proportion of TNF-a transcripts that associate with polysomes, suggesting that TIA-1 normally functions as a translational silencer. TIA-1 does not appear to regulate the production of interleukin 1b, granulocyte±macrophage colony-stimulating factor or interferon g, indicating that its effects are, at least partially, transcript speci®c. Mice lacking TIA-1 are hypersensitive to the toxic effects of LPS, indicating that this translational control pathway may regulate the organismal response to microbial stress.

Receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) is often followed by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca(2+) sensor and CRACM1 (Orai1; ) as the putative store-operated Ca(2+) channel. Overexpression of both proteins is required to reconstitute CRAC currents (I(CRAC); ). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na(+), Ba(2+), and Sr(2+) permeation relative to Ca(2+). Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca(2+) binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca(2+) ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca(2+)-selective CRAC-channel pore.

STIM1 in the endoplasmic reticulum and CRACM1 in the plasma membrane are essential molecular components for controlling the store-operated CRAC current. CRACM1 proteins multimerize and bind STIM1, and the combined overexpression of STIM1 and CRACM1 reconstitutes amplified CRAC currents. Mutations in CRACM1 determine the selectivity of CRAC currents, demonstrating that CRACM1 forms the CRAC channel's ion-selective pore, but the CRACM1 homologs CRACM2 and CRACM3 are less well characterized. Here, we show that both CRACM2 and CRACM3, when overexpressed in HEK293 cells stably expressing STIM1, potentiate I(CRAC) to current amplitudes 15-20 times larger than native I(CRAC). A nonconducting mutation of CRACM1 (E106Q) acts as a dominant negative for all three CRACM homologs, suggesting that they can form heteromultimeric channel complexes. All three CRACM homologs exhibit distinct properties in terms of selectivity for Ca(2+) and Na(+), differential pharmacological effects in response to 2-APB, and strikingly different feedback regulation by intracellular Ca(2+). Each of the CRAC channel proteins' specific functional features and the potential heteromerization provide for flexibility in shaping Ca(2+) signals, and their characteristic biophysical and pharmacological properties will aid in identifying CRAC-channel species in native cells that express them.

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 ...

The melastatin-related transient receptor potential channel TRPM2 is a plasma membrane Ca2+-permeable cation channel that is activated by intracellular adenosine diphosphoribose (ADPR) binding to the channel's enzymatic Nudix domain. Channel activity is also seen with nicotinamide dinucleotide (NAD+) and hydrogen peroxide (H2O2), but their mechanisms of action remain unknown. Here, we identify cyclic adenosine diphosphoribose (cADPR) as an agonist of TRPM2 with dual activity: at concentrations above 100 microM, cADPR can gate the channel by itself, whereas lower concentrations of 10 microM have a potentiating effect that enables ADPR to gate the channel at nanomolar concentrations. ADPR's breakdown product adenosine monophosphate (AMP) specifically inhibits ADPR, but not cADPR-mediated gating of TRPM2, whereas the cADPR antagonist 8-Br-cADPR exhibits the reverse block specificity. Our results establish TRPM2 as a coincidence detector for ADPR and cADPR signaling and provide a functional context for cADPR as a second messenger for Ca2+ influx.

Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger involved in the regulation of cell surface-associated traits and persistence. We have determined the crystal structure of PleD from Caulobacter crescentus, a response regulator with a diguanylate cyclase (DGC) domain, in its activated form. The BeF(3)(-) modification of its receiver domain causes rearrangement with respect to an adaptor domain, which, in turn, promotes dimer formation, allowing for the efficient encounter of two symmetric catalytic domains. The substrate analog GTPalphaS and two putative cations are bound to the active sites in a manner similar to adenylate cyclases, suggesting an analogous two-metal catalytic mechanism. An allosteric c-di-GMP-binding mode that crosslinks DGC and an adaptor domain had been identified before. Here, a second mode is observed that crosslinks the DGC domains within a PleD dimer. Both modes cause noncompetitive product inhibition by domain immobilization.

2-Aminoethoxydiphenyl borate (2-APB) has emerged as a useful pharmacological tool in the study of store-operated Ca 2+ entry (SOCE). It has been shown to potentiate store-operated Ca 2+ release-activated Ca 2+ (CRAC) currents at low micromolar concentrations and to inhibit them at higher concentrations. Initial experiments with the three CRAC channel subtypes CRACM1, CRACM2 and CRACM3 have indicated that they might be differentially affected by 2-APB. We now present a thorough pharmacological profile of 2-APB and report that it can activate CRACM3 channels in a store-independent manner without the requirement of STIM1, whereas CRACM2 by itself is completely unresponsive to 2-APB and CRACM1 is only very weakly activated. However, when coexpressed with STIM1 and activated via store depletion, CRACM1 and CRACM2 are facilitated at low 2-APB concentrations and inhibited at higher concentrations, while CRACM3 only exhibits potentiated currents. Consistently, the 2-APB-induced CRAC currents exhibit altered selectivities that are characterized by a leftward shift in reversal potential and the emergence of large outward currents that are carried by normally impermeant monovalent cations such as Cs + or K + . These results suggest that 2-APB has agonistic and antagonistic modes of action on CRAC channels, acting at the channel level as a store-independent and direct gating agonist for CRACM3 and a potentiating agonist for CRACM1 and CRACM2 following store-operated and STIM1-dependent activation. The inhibition of CRACM1 channels by high concentrations of 2-APB appears to involve a direct block at the channel level and an additional uncoupling of STIM1 and CRACM1, since the compound reversed the store-dependent multimerization of STIM1. Finally, we demonstrate that single-point mutations of critical amino acids in the selectivity filter of the CRACM1 pore (E106D and E190A) enable 2-APB to gate CRACM1 in a STIM1-independent manner, suggesting that 2-APB facilitates CRAC channels by altering the pore architecture.