Extracellular cysteines C226 and C232 mediate hydrogen sulfide-dependent inhibition of Orai3-mediated store-operated calcium entry.

The gaseous signaling molecule hydrogen sulfide (H2S) physiologically regulates store-operated Ca2+ entry (SOCE). The SOCE machinery consists of the plasma membrane-localized Orai channels (Orai1-3) and endoplasmic reticulum-localized stromal interaction molecule (STIM)1 and STIM2 proteins. H2S inhibits Orai3- but not Orai1- or Orai2-mediated SOCE. The current objective was to define the mechanism by which H2S selectively modifies Orai3. We measured SOCE and STIM1/Orai3 dynamics and interactions in HEK293 cells exogenously expressing fluorescently tagged human STIM1 and Orai3 in the presence and absence of the H2S donor GYY4137. Two cysteines (C226 and C232) are present in Orai3 that are absent in the Orai1 and Orai2. When we mutated either of these cysteines to serine, alone or in combination, SOCE inhibition by H2S was abolished. We also established that inhibition was dependent on an interaction with STIM1. To further define the effects of H2S on STIM1/Orai3 interaction, we performed a series of fluorescence recovery after photobleaching (FRAP), colocalization, and fluorescence resonance energy transfer (FRET) experiments. Treatment with H2S did not affect the mobility of Orai3 in the membrane, nor did it influence STIM1/Orai3 puncta formation or STIM1-Orai3 protein-protein interactions. These data support a model in which H2S modification of Orai3 at cysteines 226 and 232 limits SOCE evoked upon store depletion and STIM1 engagement, by a mechanism independent of the interaction between Orai3 and STIM1.