Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1)

Abstract

Store-operated Ca(2+) entry depends critically on physical interactions of the endoplasmic reticulum (ER) Ca(2+) sensor stromal interaction molecule 1 (STIM1) and the Ca(2+) release-activated Ca(2+) (CRAC) channel protein Orai1. Recent studies support a diffusion-trap mechanism in which ER Ca(2+) depletion causes STIM1 to accumulate at ER-plasma membrane (PM) junctions, where it binds to Orai1, trapping and activating mobile CRAC channels in the overlying PM. To determine the stoichiometric requirements for CRAC channel trapping and activation, we expressed mCherry-STIM1 and Orai1-GFP at varying ratios in HEK cells and quantified CRAC current (I(CRAC)) activation and the STIM1:Orai1 ratio at ER-PM junctions after store depletion. By competing for a limited amount of STIM1, high levels of Orai1 reduced the junctional STIM1:Orai1 ratio to a lower limit of 0.3-0.6, indicating that binding of one to two STIM1s is sufficient to immobilize the tetrameric CRAC channel at ER-PM junctions. In cells expressing a constant amount of STIM1, CRAC current was a highly nonlinear bell-shaped function of Orai1 expression and the minimum stoichiometry for channel trapping failed to evoke significant activation. Peak current occurred at a ratio of ∼2 STIM1:Orai1, suggesting that maximal CRAC channel activity requires binding of eight STIM1s to each channel. Further increases in Orai1 caused channel activity and fast Ca(2+)-dependent inactivation to decline in parallel. The data are well described by a model in which STIM1 binds to Orai1 with negative cooperativity and channels open with positive cooperativity as a result of stabilization of the open state by STIM1.

Fig. 1.

Minimum STIM:Orai stoichiometry for CRAC channel trapping at ER-PM junctions. (A) Experimental design. At low Orai expression, STIM is in excess and saturates CRAC channel binding sites at ER-PM junctions after store depletion (Upper); when Orai is in excess, it reduces the number of STIMs bound per channel to the minimum necessary for trapping (Lower). (B) Confocal images of the footprint of HEK 293 cells expressing mCh-GFP calibrator or Orai-GFP and mCh-STIM. After store depletion with 1 μM TG, Orai and STIM redistribute into colocalized puncta but mCh-GFP calibrator maintains a reticular pattern. (C) Pseudocolor image of mCh:GFP ratios in the cells from B. The STIM:Orai ratio in puncta is 0.48 ± 0.14 (mean ± SD) relative to the mCh:GFP ratio of 1.0 ± 0.38 (mean ± SD) in the calibrator cells. (D) STIM:Orai ratios in puncta (means ± SD) from single cells as a function of the mean peripheral Orai:STIM ratio measured at the cell footprint. mCh-STIM expression was allowed to vary (red dots) or was held constant (0.21 ± 0.01 a.u.; black dots). The maximum estimated error from free Orai background is indicated by the open symbols.

Fig. 2.

CRAC channel activity is a highly nonlinear function of Orai expression. (A) I_CRAC as a function of Orai-GFP expression in single cells. Each point is the current evoked by a brief hyperpolarization to −100 mV, normalized for cell capacitance (Methods). Cells expressed roughly the same level of mCh-STIM (0.21 ± 0.01 a.u., n = 37 cells) and varying amounts of Orai-GFP. In the range of Orai expression where I_CRAC declined sharply, fluorescence measurements in a subset of cells (open symbols) indicated a peripheral Orai:STIM ratio of 0.55 ± 0.02 (mean ± SEM), corresponding to 1.82 ± 0.06 STIMs per Orai subunit. (B) Reduced level of STIM lowers the threshold for Orai-induced suppression of SOCE. After store depletion, Ca²⁺ influx rates [d(F₃₅₀/F₃₈₀)/dt] were measured on Ca²⁺ readdition (Fig. S5) in cells with moderate (0.22 ± 0.01 a.u., n = 68; circles) or low (0.10 ± 0.01 a.u., n = 48; triangles) levels of mCh-STIM. Each point is the mean ± SEM of the Ca²⁺ influx rate for 4–12 cells, averaged in bins of 0.2 a.u. of Orai expression.

Fig. 3.

Rapid CDI is a highly nonlinear function of Orai expression. Data are from the cells of Fig. 2A. (A) Inactivation during hyperpolarizing steps is progressively replaced by potentiation in cells expressing increasing amounts of Orai. Each trace is the response to a step from +30 mV to −120 mV in 20 mM Ca²⁺. (B) Proportion of current remaining at the end of the hyperpolarization, quantified as the current at 195 ms (I₁₉₅) relative to that at 3 ms (I₃), as a function of Orai expression in single cells. (C) At high levels of Orai, the loss of CDI and appearance of potentiation occur in parallel with the increasing suppression of CRAC channel activity. The data in Fig. 2A are reproduced with colors indicating the current remaining at the end of the pulse.

Fig. 4.

Equilibrium model for CRAC channel gating by STIM. (A) Modified Monod–Wyman–Changeux scheme in which closed (Left) and open (Right) channel states each have four STIM binding sites, with bound sites indicated in black. Equilibrium constants for each transition are shown. K_a, STIM association constant; L, opening equilibrium constant; f, opening cooperativity factor; a, binding cooperativity factor. (B) Best fit of the model to the I_CRAC vs. Orai data of Fig. 2A, with K_a = 100, L = 10⁻⁴, f = 14.2, a = 0.5, and S_total = 3.2. (C) Model predictions superimposed on the data of Fig. 1D, assuming that channel trapping in puncta requires binding of one (blue) or two (green) STIMs to a single site. (D) Predictions of open-channel state occupancies and free STIM concentration as a function of total Orai expression. (E) Composition of the open-channel population as a function of Orai expression. In the range of moderate Orai (3–7 a.u.) in which CDI transitions to potentiation (Fig. 3C), OS₃ and OS₂ dominate over OS₄.