Hydrogen Sulfide inhibits store‐operated calcium influx by selectively targeting STIM1‐Orai3 interactions.

Hydrogen sulfide (H 2 S) is an endogenous signaling molecule that posttranslationally modifies cysteine residues on target proteins involved in regulating the physiology and pathophysiology of neurodegeneration, blood pressure, metabolism, and inflammation. We recently determined that H 2 S impinges on Ca 2+ signaling‐regulated processes by inhibiting store operated Ca 2+ entry (SOCE). The SOCE machinery consists of the plasma membrane‐localized Orai channels and ER‐localized STIM proteins. Interestingly, H 2 S inhibits SOCE mediated by STIM1‐Orai3 and not STIM1‐Orai1 or Orai2 interactions. The current study tested the hypothesis that H 2 S achieves selectively and disrupts STIM1‐Orai3 interactions by targeting cysteines unique to Orai3. The approach was to transiently transfect either CFP‐ or mCherry‐tagged Orai3 into HEK293 cells stably expressing STIM1‐YFP and measure SOCE by Ca 2+ imaging. Two cysteines (C226 and C232) are present in Orai3 that are absent in the Orai1 and Orai2 sequences. Mutation of either of these cysteines to serine, alone or in combination, abolished SOCE inhibition by the H 2 S donor GYY4137. Residues C226 and C232 in Orai3 are predicted to form an extracellular intermolecular disulfide. To determine the requirement for disulfide formation, cells were pretreated with DTT. When performed prior to GYY4137 treatment, disulfide reduction also abolished inhibition of SOCE by GYY4137. SOCE is activated upon ER Ca 2+ store depletion when STIM interacts with and gates open Orai channels. We postulated that H 2 S inhibited SOCE by inhibiting STIM1‐Orai3 interactions. To test this, cells were treated with GYY4137 both before and after store depletion. GYY4137 treatment decreased SOCE only when cells were pretreated with GYY4137. These data suggest that H 2 S modification of Orai3 inhibits the initiation of STIM1‐Orai3 interactions but cannot disrupt them once formed. We next visualized the interaction between STIM1‐YFP and Orai3‐mCherry using confocal microscopy and measured the rate of YFP‐mCherry puncta formation upon store depletion. Pre‐treatment with GYY4137 caused a decrease in puncta formation rate. Taken together, these data are consistent with a model in which H 2 S impairs SOCE by modifying a disulfide bridge on Orai3 resulting in decreased STIM1‐Orai3 interactions. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .