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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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SOCE in HSG cells is determined by Orai1, TRPC1, and STIM1.(A) [Ca2+]i measurements in fura-2 loaded HSG cells or (B) whole cell patch clamp recordings (see Material and Methods for details) in HSG cells treated as indicated to knock down either Orai1, STIM1, or TRPC1. Tg and Ca2+ were added as indicated. Right panel in (A) shows the average data for internal Ca2+ release (fluorescence increase prior to Ca2+ addition) and Ca2+ influx (fluorescence increase after Ca2+ addition; in each case resting fluorescence was subtracted from peak 340/380 ratio). ** indicate values that are significantly different (p<0.01) from the respective control value (black bars indicate values obtained in control cells). Data represent results from six different experiments and include at least 50–150 cells for each condition (see Figure S1 for additional data on TRPC1 knockdown under these conditions and the sensitivity of Tg-stimulated Ca2+ entry to Gd3+ and 2APB). (B) Left panel shows development of currents in cells following inclusion of Tg in normal external solution (arrow, traces depict currents measured at −80 mV; composition of the solutions used here and in the following experiments are provided in Materials and Methods). Right panel shows I–V relationship of the maximum currents recorded under the various conditions (note that currents are not detected without Tg stimulation of cells).
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pbio-1001025-g001: SOCE in HSG cells is determined by Orai1, TRPC1, and STIM1.(A) [Ca2+]i measurements in fura-2 loaded HSG cells or (B) whole cell patch clamp recordings (see Material and Methods for details) in HSG cells treated as indicated to knock down either Orai1, STIM1, or TRPC1. Tg and Ca2+ were added as indicated. Right panel in (A) shows the average data for internal Ca2+ release (fluorescence increase prior to Ca2+ addition) and Ca2+ influx (fluorescence increase after Ca2+ addition; in each case resting fluorescence was subtracted from peak 340/380 ratio). ** indicate values that are significantly different (p<0.01) from the respective control value (black bars indicate values obtained in control cells). Data represent results from six different experiments and include at least 50–150 cells for each condition (see Figure S1 for additional data on TRPC1 knockdown under these conditions and the sensitivity of Tg-stimulated Ca2+ entry to Gd3+ and 2APB). (B) Left panel shows development of currents in cells following inclusion of Tg in normal external solution (arrow, traces depict currents measured at −80 mV; composition of the solutions used here and in the following experiments are provided in Materials and Methods). Right panel shows I–V relationship of the maximum currents recorded under the various conditions (note that currents are not detected without Tg stimulation of cells).

Mentions: Compared to SOCE in control HSG cells (transfected with vector or scrambled siRNA; black traces in Figure 1), knockdown of endogenous Orai1, STIM1, or TRPC1 attenuated thapsigargin (Tg)-stimulated Ca2+ influx by >90%, >80%, or >60%, respectively (Figure 1A). These conditions did not significantly affect internal Ca2+ release. Western blots (Figure S1A) demonstrate the effectiveness of TRPC1 knockdown in these cells. Ca2+ entry induced by Tg treatment of HSG cells was blocked by 1 µM Gd3+ and 20 µM 2APB (Figure S1B). Further, expression of TRPC1, TRPC1+STIM1, Orai1+STIM1, or TRPC1+STIM1+Orai1 increased Tg-stimulated Ca2+ entry (Figure S1G), which was also blocked by 1 µM Gd3+ and 20 µM 2APB (Figure S1C–F). Together, these data are consistent with our previous studies [42] that Orai1, STIM1, and TRPC1 contribute to endogenous SOCE in HSG cells. Additionally, the contributions of TRPC1, STIM1, and Orai1 to SOCE were not dependent on the level of stimulation (Figure S2). The relative decrease in SOCE induced by individual knockdown of the three proteins was similar in cells stimulated with 100 µM carbachol (CCh, a maximal stimulatory concentration) or 1 µM CCh (submaximal stimulatory concentration).


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)

SOCE in HSG cells is determined by Orai1, TRPC1, and STIM1.(A) [Ca2+]i measurements in fura-2 loaded HSG cells or (B) whole cell patch clamp recordings (see Material and Methods for details) in HSG cells treated as indicated to knock down either Orai1, STIM1, or TRPC1. Tg and Ca2+ were added as indicated. Right panel in (A) shows the average data for internal Ca2+ release (fluorescence increase prior to Ca2+ addition) and Ca2+ influx (fluorescence increase after Ca2+ addition; in each case resting fluorescence was subtracted from peak 340/380 ratio). ** indicate values that are significantly different (p<0.01) from the respective control value (black bars indicate values obtained in control cells). Data represent results from six different experiments and include at least 50–150 cells for each condition (see Figure S1 for additional data on TRPC1 knockdown under these conditions and the sensitivity of Tg-stimulated Ca2+ entry to Gd3+ and 2APB). (B) Left panel shows development of currents in cells following inclusion of Tg in normal external solution (arrow, traces depict currents measured at −80 mV; composition of the solutions used here and in the following experiments are provided in Materials and Methods). Right panel shows I–V relationship of the maximum currents recorded under the various conditions (note that currents are not detected without Tg stimulation of cells).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3050638&req=5

pbio-1001025-g001: SOCE in HSG cells is determined by Orai1, TRPC1, and STIM1.(A) [Ca2+]i measurements in fura-2 loaded HSG cells or (B) whole cell patch clamp recordings (see Material and Methods for details) in HSG cells treated as indicated to knock down either Orai1, STIM1, or TRPC1. Tg and Ca2+ were added as indicated. Right panel in (A) shows the average data for internal Ca2+ release (fluorescence increase prior to Ca2+ addition) and Ca2+ influx (fluorescence increase after Ca2+ addition; in each case resting fluorescence was subtracted from peak 340/380 ratio). ** indicate values that are significantly different (p<0.01) from the respective control value (black bars indicate values obtained in control cells). Data represent results from six different experiments and include at least 50–150 cells for each condition (see Figure S1 for additional data on TRPC1 knockdown under these conditions and the sensitivity of Tg-stimulated Ca2+ entry to Gd3+ and 2APB). (B) Left panel shows development of currents in cells following inclusion of Tg in normal external solution (arrow, traces depict currents measured at −80 mV; composition of the solutions used here and in the following experiments are provided in Materials and Methods). Right panel shows I–V relationship of the maximum currents recorded under the various conditions (note that currents are not detected without Tg stimulation of cells).
Mentions: Compared to SOCE in control HSG cells (transfected with vector or scrambled siRNA; black traces in Figure 1), knockdown of endogenous Orai1, STIM1, or TRPC1 attenuated thapsigargin (Tg)-stimulated Ca2+ influx by >90%, >80%, or >60%, respectively (Figure 1A). These conditions did not significantly affect internal Ca2+ release. Western blots (Figure S1A) demonstrate the effectiveness of TRPC1 knockdown in these cells. Ca2+ entry induced by Tg treatment of HSG cells was blocked by 1 µM Gd3+ and 20 µM 2APB (Figure S1B). Further, expression of TRPC1, TRPC1+STIM1, Orai1+STIM1, or TRPC1+STIM1+Orai1 increased Tg-stimulated Ca2+ entry (Figure S1G), which was also blocked by 1 µM Gd3+ and 20 µM 2APB (Figure S1C–F). Together, these data are consistent with our previous studies [42] that Orai1, STIM1, and TRPC1 contribute to endogenous SOCE in HSG cells. Additionally, the contributions of TRPC1, STIM1, and Orai1 to SOCE were not dependent on the level of stimulation (Figure S2). The relative decrease in SOCE induced by individual knockdown of the three proteins was similar in cells stimulated with 100 µM carbachol (CCh, a maximal stimulatory concentration) or 1 µM CCh (submaximal stimulatory concentration).

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