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.Store-operated calcium entry represents a key mechanism by which cells generate Ca 2ϩ signals and maintain Ca 2ϩ homeostasis by replacing Ca 2ϩ lost from the endoplasmic reticulum (ER) 2 with Ca 2ϩ that enters the cytoplasm through plasma membrane channels. The Ca 2ϩ release-activated Ca 2ϩ (CRAC) channel is a prototypical store-operated calcium entry channel whose essential components are STIM1, the Ca 2ϩ sensor of the ER, and Orai1, the CRAC channel pore-forming subunit. STIM1 is a single-pass ER membrane protein with several functional domains, including three coiled-coil domains (CC1, CC2, and CC3) facing the cytosol, with CC2 and CC3 forming part of a minimal CRAC channel activation domain called CAD (1) or STIM-Orai activating region (SOAR) (2) (spanning residues 339 -448 in hSTIM1, referred to hereafter only as CAD). Each Orai1 subunit has four transmembrane segments, with the N and C termini of the protein facing the cytosol, and Orai1 channels are assembled from hexamers of Orai1 subunits. The activation of CRAC channels starts when depletion of calcium from the ER causes rearrangement of STIM1 and unmasking of CAD (3-8). When exposed, CAD binds to a site in the Orai1 N terminus (N-terminal binding domain (NBD), residues 74 -87) and to a second site in the Orai1 C terminus (C-terminal binding domain (CBD), residues 267-292) (1, 9). This interaction results in clustering of STIM-Orai1 at ER-PM (plasma membrane) junctions and in pore opening of Orai1 channels, which mediate calcium influx into cells (10 -17). We and others have shown recently that STIM1 binding to both the Orai1 NBD and CBD is critical for channel activation and that binding of STIM1 at these N-and C-terminal domains of Orai1 likely induces rearrangements in proximal membrane segments to open the channel (18 -20). Whether the Orai1 NBD and CBD contribute to STIM1 binding independently or through a more complex manner is unknown.In this study, we determine the relative contribution of the Orai1 NBD and CBD to interaction with STIM1 by studying the effect of cycling mutations between...

Sequential Steps of CRAC Channel Activation

“…Under basal conditions, before addition of 2-APB, EGFP-S1C fluorescence was found almost exclusively on the plasma membrane in cells expressing wt Orai1 and the addition of 2-APB did not change this distribution (Figure 4A). This is consistent with the high affinity and maximal degree of binding of SOAR to the wt channel (McNally et al, 2013; Palty and Isacoff, 2016; Park et al, 2009; Yuan et al, 2009). In cells co-expressing mCherry-Orai1 K85E, EGFP-S1C was found in both the plasma membrane and cytosol and this distribution was not affected by 2-APB (Figure 4B).…”

“…This suggests that the mutant SOAR binds to an interface in Orai1 that is exposed in the partially activated state of the channel. SOAR has been shown to interact with two sites in Orai1, a weak interaction with the N terminal region and a strong interaction with the C terminal region (Derler et al, 2013; McNally et al, 2013; Palty and Isacoff, 2016; Park et al, 2009; Yuan et al, 2009; Zhou et al, 2010). To study the contributions of the N and C terminal regions of Orai1 to SOAR interaction in the partially activated state, we separately examined key mutations in the Orai1 N terminal region (K85E) and C terminal region (L273S) that diminish or prevent, respectively, binding of soluble SOAR fragments to the resting channel(Lis et al, 2010; Muik et al, 2008; Navarro-Borelly et al, 2008).…”

“…Activated STIM1 molecules form oligomers and translocate to cortical regions of the ER that are in close proximity to the plasma membrane(Lioudyno et al, 2008; Luik et al, 2006, 2008; Wu et al, 2006; Xu et al, 2006). In these ER-PM microdomains, STIM1 binds to Orai1 through cooperative interactions with the N and C termini regions of Orai1, to gate the channel open(Lis et al, 2010; McNally et al, 2013; Palty and Isacoff, 2016; Palty et al, 2015; Park et al, 2009; Zheng et al, 2013; Zhou et al, 2010). The process of coupling STIM1 binding to induction of channel opening is, however, not well understood.…”

The STIM-Orai Pathway: Orai, the Pore-Forming Subunit of the CRAC Channel

“…The N-terminal region of Orai1 spanning residues 73–91 is essential for STIM-dependent activation of the channel (Li et al 2007; McNally et al 2013; Derler et al 2013; Zheng et al 2013; Palty and Isacoff 2016). Some specific residues that contribute to gating have been mapped in mutational studies (Lis et al 2010; Derler et al 2013; Gudlur et al 2014; Zhou et al 2016).…”

Calsequestrin-1 Regulates Store-Operated Ca2+ Entry by Inhibiting STIM1 Aggregation

“…It triggers SOCE by sensing ER store depletion, translocating as oligomers to an ER membrane compartment closely juxtaposed to the adjacent PM, forming STIM1 clusters called ‘puncta', and binding with Orai1 [4,5,6]. By a direct protein-protein interaction, the STIM1-Orai1 pathway enables Orai1 gating and Ca 2+ influx through such an effectively demarcated microdomain for Ca 2+ signaling [7,8,9]. …”