Synergistic stabilization by nitrosoglutathione-induced thiol modifications in the stromal interaction molecule-2 luminal domain suppresses basal and store operated calcium entry.

Stromal interaction molecule-1 and -2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca) sensing proteins that, together with Orai1-composed Ca channels on the plasma membrane (PM), regulate intracellular Ca levels. Recent evidence suggests that S-nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca sensing region in a Cys-specific manner. We uncovered a remarkable synergism in this stabilization involving the three luminal Cys of STIM2, which is unique to this paralog. S-Nitrosylation causes structural perturbations that converge on the face of the EF-hand and sterile α motif (EF-SAM) domain, implicated in unfolding-coupled activation. In HEK293T cells, enhanced free basal cytosolic Ca and SOCE mediated by STIM2 overexpression could be attenuated by GSNO or mutation of the modifiable Cys located in the luminal domain. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly and cooperatively affect basal Ca and SOCE regulation.