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Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions.

ABSTRACT: 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(???EE???). 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 NF?B 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.

SUBMITTER: Cheng KT

PROVIDER: S-EPMC3050638 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions.

Cheng Kwong Tai KT Liu Xibao X Ong Hwei Ling HL Swaim William W Ambudkar Indu S IS

PLoS biology 20110308 3

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 ...[more]

PMID: 21408196

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Project description:Regulation of critical cellular functions, including Ca(2+)-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca(2+) signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca(2+)](i) due to intracellular Ca(2+) release and Ca(2+) influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca(2+)](i) increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca(2+)](i) increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca(2+) entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca(2+) store depletion in these cells, it is not clear how the cells decode individual Ca(2+) signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca(2+)](i) signals and activation of NFAT in single cells. We report that Orai1-mediated Ca(2+) entry generates [Ca(2+)](i) oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca(2+) entry generates sustained [Ca(2+)](i) elevation at high [CCh] and contributes to frequency of [Ca(2+)](i) oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca(2+) dependent gene expression pathways, consistent with the different patterns of [Ca(2+)](i) signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display "all-or-none" activation that is exclusively driven by local [Ca(2+)](i) generated by Orai1, independent of global [Ca(2+)](i) changes or TRPC1-mediated Ca(2+) entry. In contrast, Ca(2+) entry via TRPC1 primarily regulates NF?B-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca(2+) signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca(2+)-dependent gene expression pathways.

Project description:There is substantial evidence indicating that disruption of Ca(2+) homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca(2+) deregulation and the Ca(2+) signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca(2+) entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca(2+) storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca(2+) storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca(2+) homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca(2+) store contributes to the disrupted Ca(2+) homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.

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Dataset Information

Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions.

Publications

Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions.

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