Cooperative Binding of Stromal Interaction Molecule 1 (STIM1) to the N and C Termini of Calcium Release-activated Calcium Modulator 1 (Orai1)

Abstract

Calcium flux through store-operated calcium entry is a central regulator of intracellular calcium signaling. The two key components of the store-operated calcium release-activated calcium channel are the Ca(2+)-sensing protein stromal interaction molecule 1 (STIM1) and the channel pore-forming protein Orai1. During store-operated calcium entry activation, calcium depletion from the endoplasmic reticulum triggers a series of conformational changes in STIM1 that unmask a minimal Orai1-activating domain (CRAC activation region (CAD)). To gate Orai1 channels, the exposed STIM1-activating domain binds to two sites in Orai1, one in the N terminus and one in the C terminus. Whether the two sites operate as distinct binding domains or cooperate in CAD binding is unknown. In this study, we show that the N and C-terminal domains of Orai1 synergistically contribute to the interaction with STIM1 and couple STIM1 binding with channel gating and modulation of ion selectivity.

Keywords: calcium channel; calcium release-activated calcium channel protein 1 (ORAI1); gating; ion channel; stromal interaction molecule 1 (STIM1).

FIGURE 1.

Untethered STIM1 fragments interact with Orai1-S or with the R429C Orai1-SS mutant. A, schematic of Orai1, the SOAR/CAD-like S1C fragment, and the Orai1-SS channels used in this work. B, summary of current densities (n = 5–8 cells, left panel) and representative plots of the current-voltage relationship (right panel) of currents recorded from cells expressing Orai1-S alone or from cells co-expressing S1C together with Orai1 or Orai1-S. pF, picofarad. C, summary of current densities (n = 5 cells, left panel) and representative plots of the current-voltage relationship (right panel) recorded from cells expressing Orai1-SS with the loss-of-function mutation R429C alone or together with WT S1C (left panel). D and E, representative fluorescence images (D) and summary of fluorescence densities (E) measured from cells expressing the WT or the R429C Orai1-SS-EGFP mutant (n = 6 regions in each). rfu, relative fluorescence units. Scale bar = 10 μm.

FIGURE 2.

Cooperative interaction between STIM1 fragments and the N and C termini of Orai1 controls channel activation. A, representative plots of the current-voltage relationship of currents recorded from cells expressing the indicated single or double Orai1-SS mutants. pF, picofarad. B, summary of current densities (n = 5–8 cells) or basal Ca²⁺ levels (n = 25–35 cells) recorded from cells expressing Orai1-SS with the indicated single or double mutations. Dashed red columns show the predicted additive effect for the double mutants. C and D, quantitation of expression levels for the Orai1 constructs used in this work. C, summary of fluorescence densities measured for the indicated Orai1-SS-EGFP mutants (n = 6 regions in each). D, representative fluorescence images of cells co-expressing the indicated Orai1-SS-EGFP mutant together with td-Tomato (as a marker for the cytosol and nucleus regions). *, p < 0.05.

FIGURE 3.

Cooperative interaction between STIM1 and the N and C termini of Orai1 controls channel activation. A, representative plots of the current-voltage relationship of currents recorded from cells expressing STIM1 with mCherry-Orai1 or with the indicated mCherry-Orai1 mutants. pF, picofarad. B, summary of current densities recorded from cells expressing STIM1 with the indicated single or double mCherry-Orai1 mutations (n = 10–15 cells). C and D, representative fluorescence images (C) and summary of fluorescence densities (D) measured from cells expressing the WT or the W76C/L276C Orai1 double mutant (n = 6 regions in each). E, representative plots of the current-voltage relationship of currents recorded from cells expressing WT Orai1-S or Orai1-S with the indicated Orai1 mutation. F, summary of current densities recorded from cells expressing Orai1-S with the indicated single or double mutations (n = 6–9 cells). G and H, representative fluorescence images (G) and summary of fluorescence densities (H) measured from cells expressing the WT Orai1-S or the W76C/L276C Orai1-S double mutant (n = 6 regions in each). Dashed red columns show the predicted additive effect for the double mutants. Scale bars = 10 μm. *, p < 0.05; **, p < 0.01.

FIGURE 4.

Cooperative interaction between STIM1 fragments and the N and C termini of Orai1 controls regulation of ion selectivity. A, average of normalized plots of the current-voltage relationship recorded in 10 mm Ca²⁺ Ringer's solution from cells expressing the indicated double or triple Orai1-SS mutants. Arrows indicate the reversal potential in each instance. B, summary of reversal potentials and current densities recorded from cells expressing the indicated Orai1-SS mutants (n = 5–6 cells). Dashed red columns show the predicted additive effect for the double mutants. *, p < 0.05; **, p < 0.01. pF, picofarad.

FIGURE 5.

Cooperative binding of S1C to the N and C termini of Orai1. A and B, representative fluorescence images (top panel) and summary of normalized intensity values (bottom panel) across the plasma membrane and intracellular regions of cells expressing EGFP-S1C together with the indicated mCherry-Orai1 mutants (n = 12–22 cells). B, quantitative summary of EGFP-S1C fraction (ΔNI = NI_PM − NI_Cyto, see inset) that is localized to the plasma membrane (PM). C, summary of FRET efficiency values measured in cells expressing EGFP-S1C together with mCherry-Orai1 or with the indicated single or double mCherry-Orai1 mutants (n = 15–35 cells). Dashed red columns show the predicted additive effect for the double mutants. *, p < 0.05; ***, p < 0.001.