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Results for “ORAI1 Protein”

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26 records · Page 2Linked to original sources
PubMed2007

CRAC channel activity in C. elegans is mediated by Orai1 and STIM1 homologues and is essential for ovulation and fertility.

The Ca(2+) release-activated Ca(2+) (CRAC) channel is a plasma membrane Ca(2+) entry pathway activated by endoplasmic reticulum (ER) Ca(2+) store depletion. STIM1 proteins function as ER Ca(2+) sensors and regulate CRAC channel activation. Recent studies have demonstrated that CRAC channels are encoded by the human Orai1 gene and a homologous Drosophila gene. C. elegans intestinal cells express a store-operated Ca(2+) channel (SOCC) regulated by STIM-1. We cloned a full-length C. elegans cDNA that encodes a 293 amino acid protein, ORAI-1, homologous to human and Drosophila Orai1 proteins. ORAI-1 GFP reporters are co-expressed with STIM-1 in the gonad and intestine. Inositol 1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signalling regulates C. elegans gonad function, fertility and rhythmic posterior body wall muscle contraction (pBoc) required for defecation. RNA interference (RNAi) silencing of orai-1 expression phenocopies stim-1 knockdown and causes sterility and prevents intestinal cell SOCC activation, but has no effect on pBoc or intestinal Ca(2+) signalling. Orai-1 RNAi suppresses pBoc defects induced by intestinal expression of a STIM-1 Ca(2+)-binding mutant, indicating that the proteins function in a common pathway. Co-expression of stim-1 and orai-1 cDNAs in HEK293 cells induces large inwardly rectifying cation currents activated by ER Ca(2+) depletion. The properties of this current recapitulate those of the native SOCC current. We conclude that C. elegans expresses bona fide CRAC channels that require the function of Orai1- and STIM1-related proteins. CRAC channels thus arose very early in animal evolution. In C. elegans, CRAC channels do not play obligate roles in all IP(3)-dependent signalling processes and ER Ca(2+) homeostasis. Instead, we suggest that CRAC channels carry out highly specialized and cell-specific signalling roles and that they may function as a failsafe mechanism to prevent Ca(2+) store depletion under pathophysiological and stress conditions.

Amino Acid Sequence↗
PubMed2006

Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai.

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) and Orai. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells. The overexpression of Stim and Orai together markedly increases CRAC current. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. Here we show that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, we show that a point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel.

Animals↗
PubMed2007

Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components.

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.

Animals↗
PubMed2006

Calcium signals mediated by STIM and Orai proteins--a new paradigm in inter-organelle communication.

In all cells Ca2+ signals are key to controlling a spectrum of cellular responses. Ca2+ signals activated by phospholipase C-coupled receptors have two components-rapid Ca2+ release from ER stores followed by slower Ca2+ entry from outside the cell. The coupling process between ER and PM to mediate this "store-operated" Ca2+ entry process has remained a molecular and mechanistic mystery. Through a combination of high throughput screening and molecular physiological approaches, the machinery and mechanism of this process have been elucidated. Two proteins are key to the coupling process. STIM1, a single spanning membrane protein with an unpaired Ca2+ binding EF-hand functions as the sensor of ER luminal Ca2+ and through redistribution in the ER transduces information directly to the PM. Orai1, a tetra-spanning PM protein, functions as the highly Ca2+ selective channel in the PM that is gated through interactions with the store-activated ER Ca2+ sensor. This molecular pas-de-deux between ER and PM components represents not only a crucial signaling pathway, but also a new paradigm in inter-organelle communication.

Amino Acid Sequence↗
PubMed2006

Store-operated Ca2+ entry: A STIMulating stOrai.

Store-operated Ca2+ entry (SOCE) mediates much of the Ca2+ entry evoked by receptors that stimulate phospholipase C. However, for 20 years, the plasma membrane Ca2+ channel and the signal linking its activation to loss of Ca2+ from the endoplasmic reticulum (ER) have eluded detection. But the search might now be over. Two proteins, STIM1 (the ER Ca2+ sensor) and Orai1 (the Ca2+ channel), have recently been identified as the missing links in SOCE.

Calcium↗
PubMed2006

Orai1 is an essential pore subunit of the CRAC channel.

Stimulation of immune cells causes depletion of Ca2+ from endoplasmic reticulum (ER) stores, thereby triggering sustained Ca2+ entry through store-operated Ca2+ release-activated Ca2+ (CRAC) channels, an essential signal for lymphocyte activation and proliferation. Recent evidence indicates that activation of CRAC current is initiated by STIM proteins, which sense ER Ca2+ levels through an EF-hand located in the ER lumen and relocalize upon store depletion into puncta closely associated with the plasma membrane. We and others recently identified Drosophila Orai and human Orai1 (also called TMEM142A) as critical components of store-operated Ca2+ entry downstream of STIM. Combined overexpression of Orai and Stim in Drosophila cells, or Orai1 and STIM1 in mammalian cells, leads to a marked increase in CRAC current. However, these experiments did not establish whether Orai is an essential intracellular link between STIM and the CRAC channel, an accessory protein in the plasma membrane, or an actual pore subunit. Here we show that Orai1 is a plasma membrane protein, and that CRAC channel function is sensitive to mutation of two conserved acidic residues in the transmembrane segments. E106D and E190Q substitutions in transmembrane helices 1 and 3, respectively, diminish Ca2+ influx, increase current carried by monovalent cations, and render the channel permeable to Cs+. These changes in ion selectivity provide strong evidence that Orai1 is a pore subunit of the CRAC channel.

Calcium↗
PubMed2026

Direct targeting of ORAI1 by ginsenoside Rg3 modulates calcium signaling and senescence-associated AMPK-NRF2 activation.

BACKGROUND: 20(S)-ginsenoside Rg3 (Rg3(S)), a major saponin derived from red ginseng, exhibits diverse biological activities, including antioxidant and anti-senescence effects. However, the direct molecular targets through which Rg3(S) regulates calcium signaling and its role at membrane contact sites during cellular senescence remain largely unknown. METHODS: Plasma membrane (PM)-endoplasmic reticulum (ER) contacts and related protein interactions were analyzed using proximity ligation assays and co-immunoprecipitation. Direct binding of Rg3(S) to ORAI1 was validated using cellular thermal shift assays and microscale thermophoresis. Molecular docking simulations followed by site-directed mutagenesis were used to define critical binding residues. Cytosolic calcium levels and cellular senescence were assessed using calcium imaging and senescence-associated β-galactosidase staining. RESULTS: Rg3(S) increased cytosolic calcium levels independently of ER calcium depletion and was accompanied by a reduction in PM-ER contacts. Rg3(S) directly bound to ORAI1 in a dose-dependent manner, identifying ORAI1 as a previously unrecognized molecular target of ginsenoside Rg3. Molecular docking revealed LYS204 and ILE229 within the extracellular loop of ORAI1 as key residues maintaining this interaction. Mutation of these residues abolished Rg3(S)-induced calcium influx, leading to impaired activation of the AMPK-NRF2 pathway and attenuation of the anti-senescence effect of Rg3(S). CONCLUSION: These findings identify ORAI1 as a key molecular mediator of ginsenoside Rg3(S)-induced calcium signaling linked to cellular senescence. By modulating PM-ER contact sites and cytosolic calcium dynamics, Rg3(S) attenuates senescence, providing new mechanistic insight into the anti-aging potential of ginseng-derived compounds beyond autophagy-centered pathways.

Calcium signaling↗
PubMed2006

STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels.

Receptor-evoked Ca2+ signalling involves Ca2+ release from the endoplasmic reticulum, followed by Ca2+ influx across the plasma membrane. Ca2+ influx is essential for many cellular functions, from secretion to transcription, and is mediated by Ca2+-release activated Ca2+ (I(crac)) channels and store-operated calcium entry (SOC) channels. Although the molecular identity and regulation of I(crac) and SOC channels have not been precisely determined, notable recent findings are the identification of STIM1, which has been indicated to regulate SOC and I(crac) channels by functioning as an endoplasmic reticulum Ca2+ sensor, and ORAI1 (ref. 7) or CRACM1 (ref. 8)--both of which may function as I(crac) channels or as an I(crac) subunit. How STIM1 activates the Ca2+ influx channels and whether STIM1 contributes to the channel pore remains unknown. Here, we identify the structural features that are essential for STIM1-dependent activation of SOC and I(crac) channels, and demonstrate that they are identical to those involved in the binding and activation of TRPC1. Notably, the cytosolic carboxyl terminus of STIM1 is sufficient to activate SOC, I(crac) and TRPC1 channels even when native STIM1 is depleted by small interfering RNA. Activity of STIM1 requires an ERM domain, which mediates the selective binding of STIM1 to TRPC1, 2 and 4, but not to TRPC3, 6 or 7, and a cationic lysine-rich region, which is essential for gating of TRPC1. Deletion of either region in the constitutively active STIM1(D76A) yields dominant-negative mutants that block native SOC channels, expressed TRPC1 in HEK293 cells and I(crac) in Jurkat cells. These observations implicate STIM1 as a key regulator of activity rather than a channel component, and reveal similar regulation of SOC, I(crac) and TRPC channel activation by STIM1.

Amino Acid Sequence↗