SOAR and the polybasic STIM1 domains gate and regulate Orai channels

Abstract

Influx of Ca(2+) through store-operated Ca(2+) channels (SOCs) is a central component of receptor-evoked Ca(2+) signals. Orai channels are SOCs that are gated by STIM1, a Ca(2+) sensor located in the ER but how it gates and regulates the Orai channels is unknown. Here, we report the molecular basis for gating of Orais by STIM1. All Orai channels are fully activated by the conserved STIM1 amino acid fragment 344-442, which we termed SOAR (the STIM1 Orai activating region). SOAR acts in combination with STIM1 (450-485) to regulate the strength of interaction with Orai1. Activation of Orai1 by SOAR recapitulates all the kinetic properties of Orai1 activation by STIM1. However, mutations of STIM1 within SOAR prevent activation of Orai1 but not co-clustering of STIM1 and Orai1 in response to Ca(2+) store depletion, indicating that STIM1-Orai1 co-clustering is not sufficient for Orai1 activation. An intact carboxy terminus alpha-helicial region of Orai is required for activation by SOAR. Deleting most of the Orai1 amino terminus impaired Orai1 activation by STIM1, but Orai1(Delta1-73) interacted with and was fully activated by SOAR. Accordingly, the characteristic inward rectification of Orai is mediated by an interaction between the polybasic STIM1 (672-685) and a Pro-rich region in the N terminus of Orai1. Hence, the essential properties of Orai1 function can be rationalized by interactions with discrete regions of STIM1.

Fig. 1. Interaction of SOAR and STIM1 fragments with Orai1 and activation of SOCs

(a) A model of STIM1 domains showing the position of SOAR. (b) The STIM1 fragments analyzed in the present work. (c) Summary of the STIM1 fragments interaction with Orai1 (first column) and activation of spontaneous Ca²⁺ influx (second column). “+” indicates activation of spontaneous Ca²⁺ influx; “−“ indicates lack of activation of spontaneous Ca²⁺ influx and inhibition of the native SOC; NE indicates no effect on spontaneous Ca²⁺ influx or SOCs; and ND indicates not determined. (d) Example traces of HEK cells transfected with EGFP or the indicated EGFP-tagged STIM1 fragments that were incubated in Ca²⁺-free and then Ca²⁺-containing media to evaluate the spontaneous Ca²⁺ influx. The cells were then treated with 25 µM CPA in Ca²⁺-free media to estimate the ER Ca²⁺ content and finally the cells were incubated with 25 µM CPA and 2 mM Ca²⁺ to measure the fully activated SOCs. The traces are representative of 4 experiments, which were used to generate the activity in the table in (c). (e) Localization of the STIM1 fragments when transfected alone (first image in each series) and when co-transfected with Orai1. Also shown is expression of STIM1(344–460) in cells treated for 72 hrs with siOrai1 and co-expression of SOAR and Orai1 in cells treated for 72 hrs with siSTIM1. All STIM1 fragments and Orai1 are N terminally tagged with EGFP and FLAG-mCherry, respectively. Scale bars are 15 microns and here and in Fig. S2, the same expression pattern was observed in 5 separate transfections and in all cells examined within each cover slip (n>20).

Fig. 2. The properties of activation of Orai1 by SOAR and the STIM1 fragments

(a) The CRAC current was measured in HEK cells expressing Orai1 and the indicated STIM1 fragments. In these and all other current measurements, Orai1 is tagged with FLAG-mCherry and the STIM1 fragments with EGFP. Preliminary experiments showed that the tags had no effect on the activity of the proteins. The cells were dialyzed with a pipette solution containing 10 mM BAPTA and external solution containing 10 mM Ca²⁺. Then the cells were perfused with divalent-free media (DFM). Here and in all Figs., dashed lines show the zero current. (b) shows the I/V for each condition at the peak current, as marked by the filled symbols in (a). (c) Shows the mean±s.e.m of 4 experiments in each condition. (d) The native STIM1 is not required for activation of Orai1 by SOAR. HEK cells treated with scrambled or siSTIM1 (the blot shows efficiency of the knock-down, full blot is shown in Fig. S7) were transfected with Orai1 alone (control) or Orai1 and SOAR and used to measure the CRAC current. The columns show the mean±s.e.m of 4 experiments. (e) Current measurement started within 10 sec of break-in using the recording conditions in (a) to evaluate the spontaneous current before substantial diffusion of BAPTA into the cells. The columns show the mean±s.e.m of 4 experiments. (f) Fast Ca²⁺-mediated inactivation of CRAC current was measured in cells expressing Orai1 and STIM1 or Orai1 and SOAR or STIM1(344–470). After maximal activation of the current, the membrane potential was stepped from a holding potential at 0 to −120 mV for 400 msec. (g) Activation of Na⁺ current by Orai1(E106D) was measured by dialyzing the cells with pipette solution containing 140 mM Cs⁺ and 10 mM BAPTA and bath solution containing 140 mM Na⁺. The cells were transfected with Orai1 and STIM1 or SOAR. The columns show the mean±s.e.m of 4 experiments.

Fig. 3. Effect of SOAR on Orai1(L273S) and (Δ1–73)Orai1

(a) The cells were co-transfected with Orai1(L273S) and SOAR. The images represent 50 analyzed cells from 3 transfections. Note that Orai1(L273S) failed to recruit SOAR to the plasma membrane. (e) Orai1(L273S) is not activated by STIM1 or SOAR. The traces are representative of 4 experiments. (c–e) CRAC current was measured in cells transfected with (Δ1–73)Orai1 and either STIM1 (black traces) or SOAR (red traces), (b) is the I/Vs and (c) is the mean±s.e.m. of 4 experiments with STIM1 and 5 experiments with SOAR.

Fig. 4. SOAR gates Orai1 but does not mediate clustering of STIM1 or co-clustering of STIM1-Orai1

(a–b) CRAC current was measured in cells transfected with Orai1 and either SOAR (black), SOAR(LQ/AA) (red), STIM1 (blue) or STIM1(LQ/AA) (green). (b) is the mean±s.e.m. of 4 experiments. In (c), cells were co-transfected with mCherry-Orai1 and EGFP-SOAR(LQ/AA). In (d), cells were transfected with STIM1(LQ/AA) alone and were untreated (upper image) or treated with 25 µM CPA in Ca²⁺- free media for 10 min. (e) shows two examples of cells co-transfected with mCherry-Orai1 and EGFP-STIM1(LQ/AA) and treated with CPA for 10 min. Arrows in (e) point to co-localized Orai1-STIM1 in puncta. Each set of images represent at least 50 analyzed cells from 2 transfections. In (f, g), cells were transfected with Orai1 and SOAR or SOAR(LQ/AA) (f) or Orai1 and STIM1 or STIM1(LQ/AA) (g) and used to IP Orai1 with anti FLAG and probe for co-IP of SOAR or STIM1 with anti-GFP. Fig. S7b shows full blot of Orai1 and Fig. S7c shows full blot of EGFP-STIM1 and EGFP-SOAR. In addition, Fig. S7c is the control for the co-IP in panels (f, g). In (h), TRPC1 was expressed in control cells alone (condition 1, black) or together with STIM1 (condition 2, magenta), STIM1(LQ/AA) (condition 3, red), or in siSTIM1-treated cells alone (condition 4, green) or together with STIM1(LQ/AA) (condition 5, blue), and the carbachol-activated TRPC1-mediated Na⁺ current was measured. The columns show the mean±s.e.m. of the % current in 4 experiments.

Fig. 5. The role of Orai1 N terminal proline-rich domain and STIM1 K-domains in Orai1 function

(a–c) CRAC current was measured in HEK cells transfected with STIM1 (black traces) or STIM1(344–460) (red traces) and Orai1 (a), Orai1(P3,5A) (b) or Orai1(P7–9A) (c). (d) Current was measured in cells transfected with wild-type STIM1 and the single proline mutants Orai1(P3A) (blue trace), Orai1(P39A) (red trace) or Orai1(P40A) (green trace). The traces represent 4 experiments with similar results. (e, f) are the mean±s.e.m. (n=4) of the current density (e) and the fold increase in current between −70 and −100 mV (f) of wild-type Orai1 and the Orai1(P3,5A) and Orai1(P39,40A) mutants. (g) Current was measured in cells transfected with Orai1(P3,5A) and either STIM1 (black trace, n=3), STIM1(ΔK) (red trace), STIM1(K684,684E) (blue trace, n=5), or STIM1(all K/E) (green trace, n=5). The mean±s.e.m. is shown in (h).