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

Abstract

Edited by Roger J. Colbran Store-operated Ca 2؉ entry (SOCE) is a ubiquitous pathway for Ca 2؉ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca 2؉-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca 2؉ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca 2؉ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca 2؉ 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 Ca 2؉ 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 specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.

Dichotomous role of the human mitochondrial Na+/Ca2+/Li+ exchanger NCLX in colorectal cancer growth and metastasis

“…The fluorescence intensity was quantified using LAS X (Leica microsystems) software. Intracellular Ca 2+ measurements Intracellular Ca 2+ imaging was performed described previously (Emrich et al, 2019;. Briefly, the cells were cultured on glass coverslips with 50-60% confluency.…”

“…The statistics were calculated by One-way ANOVA followed by post-hock Tukey's test except for B, D, and F where paired t-test was performed, *p≥0.05, **p≥0.01, ***p≥0.001 Wigerup, C., Påhlman, S., & Bexell, D. (2016) 7, 939-946. doi:10.1038/cdd.2009.16 Zhang, X., Pathak, T., Yoast, R., Emrich, S., Xin, P., Nwokonko, R. M., . .…”

Omnitemporal choreographies of IP3R and all five STIM/Orai underlie the complexity of mammalian Ca2+signaling

“…Next, we determined the functional significance of the N-terminal versus the C-terminal domains of each STIM isoform in regulating Ca 2+ oscillations by utilizing two chimeric constructs that we recently developed 40 . The first chimera consists of the N-terminal and transmembrane domains of STIM1 combined with the C-terminus of STIM2 (tk-S1N-S2C) while the second consists of the N-terminal and transmembrane domains of STIM2 combined with the C-terminus of STIM1 (tk-S2N-S1C).…”

“…This is consistent with the known properties of NFAT1, which requires robust Ca 2+ concentrations for activation and with STIM1 function described herein as the exclusive driver of Ca 2+ plateaus. We recently revealed that coordination between endogenous STIM1 and STIM2 plays a critical role in mediating SOCE in the colorectal cancer cell line HCT116 40 . Further, recent studies presented evidence that STIM2 can serve as an enhancer of receptor-activated Ca 2+ signaling by recruiting STIM1 to Orai1 channels under conditions of modest store depletion 33,37,38 , in agreement with our findings.…”

Knockout of stim2a Increases Calcium Oscillations in Neurons and Induces Hyperactive-Like Phenotype in Zebrafish Larvae

“…We observed an increase in oscillation frequency but a decrease in the average amplitude of these oscillations in neurons in the optic tectum in stim2a −/− zebrafish compared with WT fish. It was shown earlier that STIM2 deletion and the subsequent SOCE disruption caused the decrease in oscillations of non-neuronal cells [ 66 , 67 ]. However, our in vivo data showed the increased Ca 2+ oscillation frequency in brain neurons stim2a −/− mutants.…”