Sequential Steps of CRAC Channel Activation

Abstract

Interaction between the endoplasmic reticulum protein STIM1 and the plasma membrane channel ORAI1 generates calcium signals that are central for diverse cellular functions. How STIM1 binds and activates ORAI1 remains poorly understood. Using electrophysiological, optical, and biochemical techniques, we examined the effects of mutations in the STIM1-ORAI1 activating region (SOAR) of STIM1. We find that SOAR mutants that are deficient in binding to resting ORAI1 channels are able to bind to and boost activation of partially activated ORAI1 channels. We further show that the STIM1 binding regions on ORAI1 undergo structural rearrangement during channel activation. The results suggest that activation of ORAI1 by SOAR occurs in multiple steps. In the first step, SOAR binds to ORAI1, partially activates the channel, and induces a rearrangement in the SOAR-binding site of ORAI1. That rearrangement of ORAI1 then permits sequential steps of SOAR binding, via distinct molecular interactions, to fully activate the channel.

Keywords: CRAC channel; ORAI1; STIM1; calcium; gating.

Figure 1. SOAR mutants 4KA or F394H interact with Orai1 channels in a state dependent manner

(A) Cartoon representation of Orai1 and S1C mutants used in this work. (B) Summary of current densities recorded from cells co-expressing the indicated mCherry-S1C mutants together with Orai1-EGFP. (C) Summary of current densities recorded from cells co-expressing the indicated mCherry-S1C construct together with Orai1-S-EGFP. Note that mutations are introduced to S1C domains but not to the S domain. (D–F) Western blot analysis of cell lysate or immunoprecipitated material (IP) prepared from cells expressing Orai1-EGFP (D), Orai1-S-EGFP (E) or the indicated Orai1-SEGFP constructs (F) together with the indicated FLAG-mCherry-S1C constructs. Agarose beads conjugated to anti-mCherry nanododies were used to immunoprecipitate mCherry tagged proteins and antibodies against Orai1, FLAG and GFP were used for protein detection. Full blots of images shown in (D–F) and statistical analysis of data shown in (B–C) are displayed in Figure S1iii and in Figure S1ii, respectively.

Figure 2. The CRAC channel modulator 2-APB facilitates interaction between mutant S1C and Orai1 channels

(A–C) Representative fluorescent images of mCherry-Orai1 and the indicated EGFP-S1C construct before and after application of 2-APB (50μM). Note that the cellular distribution of EGFP-S1C mutants is changed following addition of 2-APB. (D) Averaged intracellular Ca²⁺ responses in cells expressing Orai1 alone (n=25) or together with the indicated S1C construct (wt n=17, 4KA n=41, F394H n=96, R429C n=46) following addition of Ca²⁺ (2mM) and 2-APB (50μM) to the extracellular solution as marked by arrows. Individual traces are shown in Figure S2.

Figure 3. Orai1 P245L interacts with S1C mutants

(A) Western blot analysis of cell lysate or IP material prepared from cells expressing the indicated EGFP-S1C constructs together with mCherry-Orai1 P245L. Agarose beads conjugated to anti-GFP antibodies were used to immunoprecipitate EGFP tagged S1C proteins and antibodies against FLAG and mCherry were used for protein detection. (B) Time course of averaged currents (left) and typical plots of the current-voltage relationship (right) of currents recorded from cells co-expressing mCherry-orai1 P245L alone or with EGFP-S1C construct. Full blots of images shown in (A) and statistical analysis of data shown in (B) are displayed in Figure S3

Figure 4. The Orai1 N and C terminal regions are required for recruitment of mutant S1C to Orai1-S channels

(A–C, Right panels) Representative fluorescent images of the indicated EGFP-S1C (wt) construct co-expressed with the indicated mCherry-Orai1 construct before and after application of 2-APB (50μM). (D-G, Left panels) The averaged ratio of the EGFP–S1C fluorescence harboring the indicated mutation ((D)- wt, (E)-4KA, (F)-F394H and (G)-R429C) and co-expressed with the indicated Orai1 construct at the plasma membrane normalized to that in the cytosol from multiple corresponding images (n=8–13 cells).

Figure 5. Interaction of SOAR with the cytosolic facing domains of Orai1

(A) Western blot analysis of cell lysate or immunoprecipitated material (IP) prepared from cells expressing the indicated FLAG-mCherry-S1C constructs together with EGFP tagged Orai1 C terminal fragment (residues 264–301). (B) Western blot analysis of cell lysate or IP material prepared from cells expressing the indicated FLAG and mCherry tagged Orai1 N terminal fragment (residues 66–91) together with the indicated EGFP-S1C constructs.

Figure 6. Conformational dynamics of the cytosolic facing C termini of Orai1

(A) Cartoon illustrations of Orai1-SNAP construct and labeling with benzylguanine (BG) derivatives. (B) Representative fluorescent images of cells expressing Orai1-SNAP and labeled with BG-Oregon-Green (OG) and BG-TMR. (C) Time course of donor (OG) and acceptor (TMR) fluorescence intensity and their ratio in cells expressing Orai1-SNAP and co-labeled with BG-OG and BG-TMR following addition of 2-APB (50μM, grey background) to the extracellular solution. Inset (Lower panel) shows the distribution of FRET change (2-APB n=21 cells, DMSO n=10, p<0.0001), estimated by calculating the difference in the acceptor/donor ration before and after addition of 2-APB or DMSO (as indicated by ΔR). (D) Time course of averaged donor and acceptor ratios as in (C) in cells expressing Orai1-SNAP alone or together with STIM1 following addition of Thapsigargin (Tg, 1μM) to the extracellular solution (Orai1 alone n=10, Orai1 + STIM1 n=8). (E) Summary of FRET efficiency values measured in cells expressing Orai1-SNAP P245L, Orai1-SNAP alone (control) or together with S1C, labeled with the SNAP dyes and subjected to treatment with DMSO or 2-APB, as indicated. Individual measurements and statistical analysis of data shown in (E) are displayed in Figure S6.