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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 KT, Liu X, Ong HL, Swaim W, Ambudkar IS - PLoS Biol. (2011)

Bottom Line: 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.By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.

View Article: PubMed Central - PubMed

Affiliation: Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, NIH, Bethesda, Maryland, United States of America.

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(⁶⁸⁴EE⁶⁸⁵). 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 NFκB 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.

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STIM1 determines clustering of TRPC1 and Orai1 in ER/PM junctional domains following Ca2+ store depletion.TIRFM was used to detect localization of TRPC1, STIM1, or Orai1 in HSG cells. (A) Localization of YFP-TRPC1 and CFP-Orai1 in HSG cells co-expressing HA-STIM1 before (−Tg) or 5 min after Ca2+-store depletion (+Tg). Left and central panels show localization of Orai1 and TRPC1 (shown by white arrows); right panels show co-localization of the proteins (Orai1-CFP, red, and YFP-TRPC1, green) with arrows showing examples of overlay (yellow) of TRPC1 and Orai1 punctae after Tg stimulation. (B) Similar experiment done with cells co-expressing Orai1-CFP, YFP-TRPC1, and mCherry-STIM1. To make the co-localization clear, in the overlay images, O1+C1 (TRPC1 is green, Orai1 is red), S1+O1 (Orai1 is green, STIM1 is red), and S1+C1 (STIM1 is red and TRPC1 is green). Arrows show examples of overlaying Orai1, TRPC1, and STIM1 punctae after Tg stimulation. (C) Localization of YFP-TRPC1 and Orai1-CFP in siSTIM1-treated HSG cells either with or without Tg (green signal indicates TRPC1 while red signal indicates Orai1). (D) Blot on left shows co-IP of endogenous TRPC1 and Orai1 in control and siSTIM1-treated cells (minus or plus Tg). Anti-Orai1 antibody was used for IP while anti-TRPC1 was used for IB. Inputs for TRPC1, STIM1, and Orai1 in each condition are shown in the lower three panels. The bar graph on the right shows quantitation of the blots from four similar experiments. ** indicates values significantly, p<0.01, different from controls, which are not different from each other. (E) TIRFM images of Orai1-CFP and YFP-TRPC1 in HSG cells expressing STIM1(684EE685). In the overlay, Orai1 is shown in red while TRPC1 is shown in green, with yellow indicating co-localization of the two proteins. (F) Tg-induced Co-IP of TRPC1, Orai1, and STIM1 in HSG cells expressing Myc-STIM1(684EE685) in resting conditions and after stimulation with Tg. Anti-TRPC1 antibody was used for IP and anti-Orai1; STIM1 or TRPC1 was used for IB, as indicated. Inputs and control blots are also shown. Quantitation of data from four similar experiments is shown by bar graph on the right, ** indicates values significant at p<0.01.
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pbio-1001025-g004: STIM1 determines clustering of TRPC1 and Orai1 in ER/PM junctional domains following Ca2+ store depletion.TIRFM was used to detect localization of TRPC1, STIM1, or Orai1 in HSG cells. (A) Localization of YFP-TRPC1 and CFP-Orai1 in HSG cells co-expressing HA-STIM1 before (−Tg) or 5 min after Ca2+-store depletion (+Tg). Left and central panels show localization of Orai1 and TRPC1 (shown by white arrows); right panels show co-localization of the proteins (Orai1-CFP, red, and YFP-TRPC1, green) with arrows showing examples of overlay (yellow) of TRPC1 and Orai1 punctae after Tg stimulation. (B) Similar experiment done with cells co-expressing Orai1-CFP, YFP-TRPC1, and mCherry-STIM1. To make the co-localization clear, in the overlay images, O1+C1 (TRPC1 is green, Orai1 is red), S1+O1 (Orai1 is green, STIM1 is red), and S1+C1 (STIM1 is red and TRPC1 is green). Arrows show examples of overlaying Orai1, TRPC1, and STIM1 punctae after Tg stimulation. (C) Localization of YFP-TRPC1 and Orai1-CFP in siSTIM1-treated HSG cells either with or without Tg (green signal indicates TRPC1 while red signal indicates Orai1). (D) Blot on left shows co-IP of endogenous TRPC1 and Orai1 in control and siSTIM1-treated cells (minus or plus Tg). Anti-Orai1 antibody was used for IP while anti-TRPC1 was used for IB. Inputs for TRPC1, STIM1, and Orai1 in each condition are shown in the lower three panels. The bar graph on the right shows quantitation of the blots from four similar experiments. ** indicates values significantly, p<0.01, different from controls, which are not different from each other. (E) TIRFM images of Orai1-CFP and YFP-TRPC1 in HSG cells expressing STIM1(684EE685). In the overlay, Orai1 is shown in red while TRPC1 is shown in green, with yellow indicating co-localization of the two proteins. (F) Tg-induced Co-IP of TRPC1, Orai1, and STIM1 in HSG cells expressing Myc-STIM1(684EE685) in resting conditions and after stimulation with Tg. Anti-TRPC1 antibody was used for IP and anti-Orai1; STIM1 or TRPC1 was used for IB, as indicated. Inputs and control blots are also shown. Quantitation of data from four similar experiments is shown by bar graph on the right, ** indicates values significant at p<0.01.

Mentions: The mechanism involved in the clustering of TRPC1 with STIM1 and Orai1 was assessed by TIRFM. Ca2+ store depletion resulted in co-localization of YFP-TRPC1 and Orai1-CFP into puncta in the sub-plasma membrane region (Figure 4A, HA-STIM1 was co-expressed in these cells). Further, STIM1 co-clustered with both the channels following Tg stimulation of the cells (Figure 4B). As has been reported for Orai1, Orai1-TRPC1 clustering also required co-expression of STIM1 (unpublished data) and was not detected in cells when endogenous STIM1 expression was knocked down (Figure 4C). More significantly, co-IP of endogenous TRPC1 and Orai1 was abolished in cells treated with siSTIM1 (Figure 4D) but not in cells expressing STIM1(685EE685) (Figure 4E,F). TRPC1 clustering was not dependent on Orai1 since co-clustering of TRPC1 with STIM1 was unaffected by knockdown of Orai1 (Figure S4, compare data in A and B). Thus, STIM1 determines TRPC1 clustering in the sub-plasma membrane region following Ca2+ store depletion, and Orai1-mediated Ca2+ entry regulates its surface expression. Based on these findings we hypothesize that TRPC1 is present in recycling vesicles that traffic in and out of the plasma membrane region. Following store depletion when STIM1 clusters in ER/PM junctional domains, it interacts with TRPC1 possibly via the ERM domain [46] and increases the retention of TRPC1-containing vesicles. Concurrently, STIM1 also recruits Orai1 into the same regions, thus bringing the two channels in close proximity to each other. Ca2+ entry via Orai1 induces fusion of TRPC1-containing vesicles to the plasma membrane followed by gating of the channel by STIM1. Further studies are required to elucidate the mechanisms involved in trafficking and plasma membrane insertion of TRPC1.


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

Cheng KT, Liu X, Ong HL, Swaim W, Ambudkar IS - PLoS Biol. (2011)

STIM1 determines clustering of TRPC1 and Orai1 in ER/PM junctional domains following Ca2+ store depletion.TIRFM was used to detect localization of TRPC1, STIM1, or Orai1 in HSG cells. (A) Localization of YFP-TRPC1 and CFP-Orai1 in HSG cells co-expressing HA-STIM1 before (−Tg) or 5 min after Ca2+-store depletion (+Tg). Left and central panels show localization of Orai1 and TRPC1 (shown by white arrows); right panels show co-localization of the proteins (Orai1-CFP, red, and YFP-TRPC1, green) with arrows showing examples of overlay (yellow) of TRPC1 and Orai1 punctae after Tg stimulation. (B) Similar experiment done with cells co-expressing Orai1-CFP, YFP-TRPC1, and mCherry-STIM1. To make the co-localization clear, in the overlay images, O1+C1 (TRPC1 is green, Orai1 is red), S1+O1 (Orai1 is green, STIM1 is red), and S1+C1 (STIM1 is red and TRPC1 is green). Arrows show examples of overlaying Orai1, TRPC1, and STIM1 punctae after Tg stimulation. (C) Localization of YFP-TRPC1 and Orai1-CFP in siSTIM1-treated HSG cells either with or without Tg (green signal indicates TRPC1 while red signal indicates Orai1). (D) Blot on left shows co-IP of endogenous TRPC1 and Orai1 in control and siSTIM1-treated cells (minus or plus Tg). Anti-Orai1 antibody was used for IP while anti-TRPC1 was used for IB. Inputs for TRPC1, STIM1, and Orai1 in each condition are shown in the lower three panels. The bar graph on the right shows quantitation of the blots from four similar experiments. ** indicates values significantly, p<0.01, different from controls, which are not different from each other. (E) TIRFM images of Orai1-CFP and YFP-TRPC1 in HSG cells expressing STIM1(684EE685). In the overlay, Orai1 is shown in red while TRPC1 is shown in green, with yellow indicating co-localization of the two proteins. (F) Tg-induced Co-IP of TRPC1, Orai1, and STIM1 in HSG cells expressing Myc-STIM1(684EE685) in resting conditions and after stimulation with Tg. Anti-TRPC1 antibody was used for IP and anti-Orai1; STIM1 or TRPC1 was used for IB, as indicated. Inputs and control blots are also shown. Quantitation of data from four similar experiments is shown by bar graph on the right, ** indicates values significant at p<0.01.
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pbio-1001025-g004: STIM1 determines clustering of TRPC1 and Orai1 in ER/PM junctional domains following Ca2+ store depletion.TIRFM was used to detect localization of TRPC1, STIM1, or Orai1 in HSG cells. (A) Localization of YFP-TRPC1 and CFP-Orai1 in HSG cells co-expressing HA-STIM1 before (−Tg) or 5 min after Ca2+-store depletion (+Tg). Left and central panels show localization of Orai1 and TRPC1 (shown by white arrows); right panels show co-localization of the proteins (Orai1-CFP, red, and YFP-TRPC1, green) with arrows showing examples of overlay (yellow) of TRPC1 and Orai1 punctae after Tg stimulation. (B) Similar experiment done with cells co-expressing Orai1-CFP, YFP-TRPC1, and mCherry-STIM1. To make the co-localization clear, in the overlay images, O1+C1 (TRPC1 is green, Orai1 is red), S1+O1 (Orai1 is green, STIM1 is red), and S1+C1 (STIM1 is red and TRPC1 is green). Arrows show examples of overlaying Orai1, TRPC1, and STIM1 punctae after Tg stimulation. (C) Localization of YFP-TRPC1 and Orai1-CFP in siSTIM1-treated HSG cells either with or without Tg (green signal indicates TRPC1 while red signal indicates Orai1). (D) Blot on left shows co-IP of endogenous TRPC1 and Orai1 in control and siSTIM1-treated cells (minus or plus Tg). Anti-Orai1 antibody was used for IP while anti-TRPC1 was used for IB. Inputs for TRPC1, STIM1, and Orai1 in each condition are shown in the lower three panels. The bar graph on the right shows quantitation of the blots from four similar experiments. ** indicates values significantly, p<0.01, different from controls, which are not different from each other. (E) TIRFM images of Orai1-CFP and YFP-TRPC1 in HSG cells expressing STIM1(684EE685). In the overlay, Orai1 is shown in red while TRPC1 is shown in green, with yellow indicating co-localization of the two proteins. (F) Tg-induced Co-IP of TRPC1, Orai1, and STIM1 in HSG cells expressing Myc-STIM1(684EE685) in resting conditions and after stimulation with Tg. Anti-TRPC1 antibody was used for IP and anti-Orai1; STIM1 or TRPC1 was used for IB, as indicated. Inputs and control blots are also shown. Quantitation of data from four similar experiments is shown by bar graph on the right, ** indicates values significant at p<0.01.
Mentions: The mechanism involved in the clustering of TRPC1 with STIM1 and Orai1 was assessed by TIRFM. Ca2+ store depletion resulted in co-localization of YFP-TRPC1 and Orai1-CFP into puncta in the sub-plasma membrane region (Figure 4A, HA-STIM1 was co-expressed in these cells). Further, STIM1 co-clustered with both the channels following Tg stimulation of the cells (Figure 4B). As has been reported for Orai1, Orai1-TRPC1 clustering also required co-expression of STIM1 (unpublished data) and was not detected in cells when endogenous STIM1 expression was knocked down (Figure 4C). More significantly, co-IP of endogenous TRPC1 and Orai1 was abolished in cells treated with siSTIM1 (Figure 4D) but not in cells expressing STIM1(685EE685) (Figure 4E,F). TRPC1 clustering was not dependent on Orai1 since co-clustering of TRPC1 with STIM1 was unaffected by knockdown of Orai1 (Figure S4, compare data in A and B). Thus, STIM1 determines TRPC1 clustering in the sub-plasma membrane region following Ca2+ store depletion, and Orai1-mediated Ca2+ entry regulates its surface expression. Based on these findings we hypothesize that TRPC1 is present in recycling vesicles that traffic in and out of the plasma membrane region. Following store depletion when STIM1 clusters in ER/PM junctional domains, it interacts with TRPC1 possibly via the ERM domain [46] and increases the retention of TRPC1-containing vesicles. Concurrently, STIM1 also recruits Orai1 into the same regions, thus bringing the two channels in close proximity to each other. Ca2+ entry via Orai1 induces fusion of TRPC1-containing vesicles to the plasma membrane followed by gating of the channel by STIM1. Further studies are required to elucidate the mechanisms involved in trafficking and plasma membrane insertion of TRPC1.

Bottom Line: 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.By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.

View Article: PubMed Central - PubMed

Affiliation: Secretory Physiology Section, Molecular Physiology and Therapeutics Branch, NIDCR, NIH, Bethesda, Maryland, United States of America.

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(⁶⁸⁴EE⁶⁸⁵). 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 NFκB 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.

Show MeSH
Related in: MedlinePlus