Cross-talk between N-terminal and C-terminal domains in stromal interaction molecule 2 (STIM2) determines enhanced STIM2 sensitivity

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 intoan active conformation in response toERCa²⁺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 colorectalHCT116cells.Weshow 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 supportitsstore- independentactivation.STIM1/STIM2chimericconstructs 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 specificSTIMisoforms, insights that are potentially useful for isoform-selective drug targeting.