Abstract
Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1-/-, STIM2-/-, and STIM1/2-/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.
Highlights
Store-operated Ca2؉ entry (SOCE) is a ubiquitous pathway for Ca2؉ influx across the plasma membrane (PM)
Western blot analysis of HEK293 STIM1Ϫ/Ϫ and STIM2Ϫ/Ϫ cell lines demonstrated no compensatory up-regulation of stromal interaction molecule 1 (STIM1) or STIM2 proteins in response to individual STIM knockout, whereas STIM1/2Ϫ/Ϫ cells were devoid of both proteins (Fig. 1, A and B)
HCT116 STIM2Ϫ/Ϫ cells showed substantial reduction in SOCE (ϳ44% of WT) despite having STIM1 protein present. These results suggest that STIM2 plays differential, cell type-specific role in supporting SOCE in response to maximal endoplasmic reticulum (ER) store depletion
Summary
Cross-talk between N-terminal and C-terminal domains in stromal interaction molecule 2 (STIM2) determines enhanced STIM2 sensitivity. High concentrations of 2-APB have been demonstrated to: 1) inhibit several constitutively active STIM-independent ORAI1 channel mutants, whereas potentiating WT ORAI3, suggesting that 2-APB acts directly on ORAI channel pore; 2) strengthen intramolecular interaction within the STIM1 C terminus, between the coiled-coil 1 (CC1) domain and SOAR1, preventing STIM1 unfolding in response to store depletion. This would result in a reduction in STIM1 oligomerization and puncta formation that would reduce functional interactions with ORAI1 (22, 26 –31). Development of 2-APB– derived analogues with increased specificity and potency for STIM2 will selectively target cellular pathways that rely on STIM2 in both healthy and disease conditions
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