Hydrogen Sulfide Inhibits Store‐Operated Calcium Entry by Targeting Orai3

Store‐operated Ca 2+ entry (SOCE) is activated in response to depletion of endoplasmic reticulum (ER) Ca 2+ stores. The molecular machinery consists of the ER‐localized stromal interaction molecule (STIM), which upon store depletion, binds to and opens the plasma membrane channel Orai. Activation of SOCE is subject to redox modifications of both STIM and Orai. The gaseous signaling molecule hydrogen sulfide (H 2 S) is a known redox modifier of ion channels, however, its potential to affect SOCE has not been explored. To study this we generated HEK293 cells stably overexpressing human STIM1 and then transiently expressed either human Orai1, 2 or 3. The STIM1 and Orai proteins were tagged with YFP and CFP respectively to identify co‐transfected cells and SOCE quantified by Ca 2+ imaging. Treatment with the H 2 S donor GYY4137 (500 μM) had no effect on SOCE mediated by Orai1 or 2 but inhibited the SOCE mediated by Orai3. It is established that H 2 S functionally affects proteins by modifying the thiol groups of reactive cysteine residues. Sequence analysis of human Orai1, 2 and 3 revealed four cysteine residues in the human and mouse Orai3 protein. Two of these (C101 and C118) are conserved in the Orai1 and Orai2 sequence; however, C226 and C232 are unique to Orai3. To assess there role in mediating H 2 S‐dependent inhibition these residues were mutated to serine. The SOCE generated by Orai3 C226S and C232S mutants was not significantly different from Orai3 wild type; however, the inhibitory effect of H 2 S was lost. These data are consistent with a model in which H 2 S inhibits SOCE mediated only by STIM1/Orai3 and not STIM1/Orai1 or STIM1/Orai2 by targeting specific cysteine residues in Orai3. Support or Funding Information DePaul‐RFUMS Collaborative Research Grant.