Stoichiometric requirements for trapping and gating of Ca ²⁺ release-activated Ca ²⁺ (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.

Mutations of the Ca2+-sensing Stromal Interaction Molecule STIM1 Regulate Ca2+ Influx by Altered Oligomerization of STIM1 and by Destabilization of the Ca2+ Channel Orai1

“…The CRAC channel states defined in this way become more stable the more STIM1 dimers participate in forming the channel, which is quantified by the cooperativity factor ␤ (16). Each of these four states contributes in a specific, predefined way to the total CRAC channel current, I CRAC ϭ 0.001⅐OS 1 ϩ 0.025⅐OS 2 ϩ 0.275⅐OS 3 ϩ 1.0⅐OS 4 , modeling the experimentally observed graded channel currents (15,16). The parameters (reaction rates and diffusion constants) of our model are shown in Table 1.…”

“…6B and parameters shown in Table 1 and assuming an optimal binding stoichiometry of eight STIM1s per tetramer of Orai1 (for details see "Experimental Procedures" and Ref. 16), the model (Fig. 6C, triangles) predicts maximal I CRAC steady state currents at a total input ratio of 7.5 STIM1:Orai1 (tetramer), with declining currents at higher Orai1 concentrations (lower S1/O1 ratios).…”

“…4B, right panel). An increased STIM1: Orai1 ratio is likely to result in a larger fraction of fully functional channels on the plasma membrane and a significant reduction in the number of silent channels formed by Orai1 tetramers with just one or two STIM1 dimers bound (16), thus possibly explaining the increased CD seen with STIM1 DQ. To also test ratio effects in a more native system, we investigated SOCE of Jurkat T cells after down-regulating Orai1 using siRNA or by recording from stably Orai1 overexpressing Jurkat T cells.…”

“…The domains of clustered STIM1/Orai1 molecules are the sites of Ca 2ϩ influx (12)(13)(14). The minimal requirement for activation are two STIM1 molecules bound per Orai1 tetramer; however, this stoichiometry results in channels with very low open probability (P o ), whereas maximal P o is achieved with eight bound STIM1 proteins per Orai1 tetramer (15,16). Failure to activate this Ca 2ϩ entry pathway either by defective STIM1 molecules or by mutations in Orai1 proteins results in severe defects in immune cell function but also in defective skin, platelet, and neuronal functions, among others (5,17,18).…”

Orai1 pore residues control CRAC channel inactivation independently of calmodulin

“…Most constructs expressed well within 24 h, but some required up to 48 h to yield currents of ideal size. STIM1-and Orai1-derived constructs were transfected in a 4:1 ratio by mass, higher than the 1:1 mass ratio used in our previous study of CDI ), because it is now recognized that maximal CDI requires a high STIM1/Orai1 ratio (Scrimgeour et al, 2009;Hoover and Lewis, 2011). In the standard experimental design, cells were transfected with mCh-STIM1 and Orai1-GFP, and were visualized with a filter cube that included a 470 ± 20-nm bandpass excitation filter, a 500-nm dichroic, and a 515 longpass emission filter.…”