Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels

Abstract

Orai1 and TRPC1 have been proposed as core components of store-operated calcium release-activated calcium (CRAC) and store-operated calcium (SOC) channels, respectively. STIM1, a Ca(2+) sensor protein in the endoplasmic reticulum, interacts with and mediates store-dependent regulation of both channels. We have previously reported that dynamic association of Orai1, TRPC1, and STIM1 is involved in activation of store-operated Ca(2+) entry (SOCE) in salivary gland cells. In this study, we have assessed the molecular basis of TRPC1-SOC channels in HEK293 cells. We report that TRPC1+STIM1-dependent SOCE requires functional Orai1. Thapsigargin stimulation of cells expressing Orai1+STIM1 increased Ca(2+) entry and activated typical I(CRAC) current. STIM1 alone did not affect SOCE, whereas expression of Orai1 induced a decrease. Expression of TRPC1 induced a small increase in SOCE, which was greatly enhanced by co-expression of STIM1. Thapsigargin stimulation of cells expressing TRPC1+STIM1 activated a non-selective cation current, I(SOC), that was blocked by 1 microm Gd(3+) and 2-APB. Knockdown of Orai1 decreased endogenous SOCE as well as SOCE with TRPC1 alone. siOrai1 also significantly reduced SOCE and I(SOC) in cells expressing TRPC1+STIM1. Expression of R91WOrai1 or E106QOrai1 induced similar attenuation of TRPC1+STIM1-dependent SOCE and I(SOC), whereas expression of Orai1 with TRPC1+STIM1 resulted in SOCE that was larger than that with Orai1+STIM1 or TRPC1+STIM1 but not additive. Additionally, Orai1, E106QOrai1, and R91WOrai1 co-immunoprecipitated with similar levels of TRPC1 and STIM1 from HEK293 cells, and endogenous TRPC1, STIM1, and Orai1 were co-immunoprecipitated from salivary glands. Together, these data demonstrate a functional requirement for Orai1 in TRPC1+STIM1-dependent SOCE.

FIGURE 1.

Endogenous Orai1 contributes to TRPC1-dependent SOCE in HEK 293 cells. Tg-stimulated SOCE or whole cell current were measured 48 h after transfection. Panels i–iv, Fura 2 measurements (panel i), average Ca²⁺ entry data (panel ii), whole cell currents at –80 mV (panel iii), and I-V curve (panel iv) in control HEK293 cells (black traces) or cells transfected with Orai1 and STIM1 (red traces). Panels v–viii, red traces, the effect of Orai1 siRNA on SOCE in control cells (panel v) and cells transfected with TRPC1 (panel vi) and TRPC1+STIM1 (panel vii); average data of Ca²⁺ influx are shown in panel viii. All traces represent average of at least 50 cells, and statistical significance is indicated by ** (p < 0.01). The effect of siOrai1 on expression of Orai1 is shown by blot (panel vi, inset). Cells were treated with siRNAs for 48 h, and cell lysate (30 μg of protein) was loaded in each lane. Lane 1, sample from cells treated with control siRNA (Orai1 in this sample is similar to that in untreated control cells, not shown); lanes 2, 3, and 4, samples from cells treated with 0.2, 0.4, or 0.8 nmol of siOrai1, respectively. Actin expression (lower blot) is not affected by siOrai1. 0.8 nmol of siOrai1 were used in all the experiments. I_SOC was detected in cells transfected with STIM1 and TRPC1 (panel ix); external application of 20 μm 2APB was indicated by the bar. I and II indicate the points represented by the IV curves shown in panel x. Panels xi and xii, I_SOC (panel xi) and I-V curves (panel xii) in cells transfected with Orai1 siRNA, STIM1, and TRPC1. All traces are representative from 4 to 10 cells each from multiple experiments (see Fig. 2, panel x, for average values).

FIGURE 2.

Functional Orai1 is required for TRPC1+STIM1-dependent SOC channel function. Panel i, Tg-stimulated SOCE was measured in Fura-2AM-loaded cells. [Ca²⁺]_i is expressed as 340/380 ratio, and traces represent average of at least 50 cells (average data from at least three individual experiments are shown in panel ii). The effect of expression of Orai1 (red traces), R91WOrai1 (blue trace), and E106QOrai1 (green trace) on SOCE in HEK293 cells expressing TRPC1+STIM1 (SOCE in control cells is shown by a black trace. SOCE in control cells expressing mutant Orais are not shown in panel i) is shown. Average data are shown by respectively colored bars in panel ii. Statistical significance is indicated by **, p < 0.01). Panel iii, the blot on the left shows co-immunoprecipitation of Orai1 (upper panels) or R91W (middle panels) and E106QOrai1 (lower panels) with STIM1 and TRPC1 from cells transfected with FLAG-tagged Orai proteins. 3 mg of cell lysates were used for IP. Similar expression levels of the Orai proteins as well as association with STIM1 or TRPC1 were detected (IP, anti-FLAG antibody; IB, immunoblot antibodies indicated in the figure. Control IP with non-transfected HEK cell lysate is shown in supplemental Fig. 2). The blot on the right shows co-immunoprecipitation of endogenous Orai1, TRPC1, and STIM1 from mouse submandibular glands (SMG). Anti-TRPC1 was used as IP antibody, and immunoblot antibodies are indicated. Tg-induced whole cell currents were recorded from cells transfected with TRPC1, STIM1 together with Orai1 (panel iv), R91WOrai1 (panel vi), or E106QOrai1 (panel viii). The respective I-V curves are shown in (panel v), (panel vii), and (panel ix). Average data from current measurements (amplitude at –80 mV) in Figs. 1 and 2 are given in panel x. Statistically significant differences are indicated by ** (p < 0.01) as is the number of the cell tested in each case (columns represent following conditions: HEK, control cells; OS, Orai1+STIM1 transfected; SC, TRPC1+STIM1 transfected; OSC, Orai1+STIM1+TRPC1 transfected; siOSC, siOrai1+STIM1+TRPC1 transfected; R91WSC, R91WOrai1+STIM1+TRPC1; E106QSC, E106QOrai1+STIM1+TRPC1).