CRACM1 Is a Plasma Membrane Protein Essential for Store-Operated Ca ²⁺ Entry

Abstract

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...

Nitric Oxide-cGMP-PKG Pathway Acts on Orai1 to Inhibit the Hypertrophy of Human Embryonic Stem Cell-Derived Cardiomyocytes

“…The sequences used were 5 0 -CGTGCACAATCTCAACTCG-3 0 (in coding region) and 5 0 -CCAGCATTGAGTGTGTACA-3 0 (in 3 0 -UTA) as described elsewhere [24,25]. Myc-Orai1 and Myc-Orai1 G98A plasmids were kind gifts from Birnbaumer L (NIH).…”

“…Orai1-specific shRNAs are also reported [24,25]. Orai1 G98A and two separate Orai1-shRNAs were individually subcloned into an adenoviral (Ad) vector to generate Ad-Orai1 G98A and Ad-Orai1-shRNAs, respectively.…”

Thapsigargin activates Ca2+ entry both by store-dependent, STIM1/Orai1-mediated, and store-independent, TRPC3/PLC/PKC-mediated pathways in human endothelial cells

“…More recently, two major SOCE components have been identified: the Stromal Interacting Molecule 1 (STIM1) [20][21][22] predominantly located in the ER membrane and that senses the luminal Ca 2+ concentration, and the Ca 2+ channel Orai1 (also known as CRACM; [23][24][25]). Upon Ca 2+ store depletion, STIM1 oligomerizes, translocates close to the plasma membrane and aggregates into punctate structures [2,21,26].…”

Dynamic Movement of the Calcium Sensor STIM1 and the Calcium Channel Orai1 in Activated T-Cells: Puncta and Distal Caps

“…STIM1 probably serves as the sensor that detects the depletion of Ca 2ϩ stores. Further RNAi screens identified Orai1 or CRACM1 as a second essential component in CRAC conductance Vig et al, 2006b;Zhang et al, 2006). Orai1 is a four-pass integral membrane protein found in the PM that forms the channel.…”

“…© 2008 by The American Society for Cell Biology CRAC conductance Vig et al, 2006b;Zhang et al, 2006). Orai1 is a four-pass integral membrane protein found in the PM that forms the channel.…”