Molecular Determinants for STIM1 Activation During Store- Operated Ca2+ Entry

Abstract

Background STIM/ORAI-mediated store-operated Ca2+ entry (SOCE) mediates a myriad of Ca2+-dependent cellular activities in mammals. Genetic defects in STIM1/ORAI1 lead to devastating severe combined immunodeficiency; whereas gain-of-function mutations in STIM1/ORAI1 are intimately associated with tubular aggregate myopathy. At molecular level, a decrease in the Ca2+ concentrations within the lumen of endoplasmic reticulum (ER) initiates multimerization of the STIM1 luminal domain to switch on the STIM1 cytoplasmic domain to engage and gate ORAI channels, thereby leading to the ultimate Ca2+ influx from the extracellular space into the cytosol. Despite tremendous progress made in dissecting functional STIM1-ORAI1 coupling, the activation mechanism of SOCE remains to be fully characterized. Objective and Methods Building upon a robust fluorescence resonance energy transfer assay designed to monitor STIM1 intramolecular autoinhibition, we aimed to systematically dissect the molecular determinants required for the activation and oligomerization of STIM1. Results Here we showed that truncation of the STIM1 luminal domain predisposes STIM1 to adopt a more active conformation. Replacement of the single transmembrane (TM) domain of STIM1 by a more rigid dimerized TM domain of glycophorin A abolished STIM1 activation. But this adverse effect could be partially reversed by disrupting the TM dimerization interface. Moreover, our study revealed regions that are important for the optimal assembly of heterooligomers composed of full-length STIM1 with its minimal STIM1-ORAI activating region, SOAR. Conclusions Our study clarifies the roles of major STIM1 functional domains in maintaining a quiescent configuration of STIM1 to prevent preactivation of SOCE.

Molecular anatomy of the early events in STIM1 activation – oligomerization or conformational change?

“…Our model is fully compatible with the findings and conclusions of the Ma study in that the reorientation of the TM segments is a key step in the early activation cascade. A more recent study from the Zhou laboratory also reached similar conclusions (Ma et al, 2017). Jing et al has identified an additional player in the control of the CC1 domain SOAR interaction.…”

“…They discovered that an ER-resident multipass transmembrane molecule they named STIMATE (also known as TMEM110) can also interact with the CC1 domain to weaken its interaction with SOAR (Jing et al, 2015). Although STIMATE appears to be an important modifier of the STIM1 activation process, it probably does not significantly change the conclusions of our study and those of Ma et al (Ma et al, 2015(Ma et al, , 2017.…”

Optogenetic engineering to probe the molecular choreography of STIM1-mediated cell signaling

“…Optogenetic mimicry of CC1-SOAR mediated autoinhibition. The interaction between CC1 and SOAR is required for STIM1 autoinhibition and to keep STIM1 inactive at the resting state 14,26,27 . We confirmed this by splitting the full-length STIM1 into two components at position 342, and appended each part with a different fluorescent protein tag ( Fig.…”

Calcium store refilling and STIM activation in STIM- and Orai-deficient cell lines

“…FRET measurements were recorded and analyzed with procedures similar to those described earlier [ 26 ]. Briefly, fluorescence imaging experiments were carried out using a ZEISS observer-Z1 microscope equipped with X-Cite® 120-Q (Lumen dynamics) light source, Semrock Bright Line filter sets (CFP (438 ± 12 Ex /482 ± 16 Em ), YFP (500 ± 12 Ex /542 ± 13.5 Em ), FRET raw (438 ± 12 Ex /542 ± 13.5 Em ), × 40 oil objective (N.A.…”