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Hydrogen sulfide inhibits Orai3‐mediated store‐operated Ca 2+ entry by targeting extracellular cysteines
Author(s) -
Fresquez Adriana Maria,
White Carl
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1007.20
Subject(s) - microbiology and biotechnology , stim1 , orai1 , endoplasmic reticulum , hek 293 cells , chemistry , extracellular , biochemistry , biology , receptor
The store operated calcium entry (SOCE) pathway is activated by interactions between the endoplasmic reticulum localized STIM1 and STIM2 proteins and the Orai 1, 2 and 3 plasmalemmal Ca 2+ channels. We recently discovered that the gaseous signaling molecule hydrogen sulfide (H 2 S) selectively inhibits SOCE mediated by STIM1 and Orai3. We showed that STIM1/Orai3 regulation has physiological importance in the innate immune system where endogenously produced H 2 S limits maximal activation of SOCE and proinflammatory cytokine production. The purpose of the current study was to define the mechanism by which H 2 S modifies STIM1/Orai3 function. It is known that H 2 S impinges on protein function by targeting disulfide bridges. Sequence analysis of human Orai1, 2 and 3 revealed two cysteines (C226 and C232) unique to the human and mouse Orai3 protein. We hypothesized that C226 and C232 are the site of action for H 2 S on Orai3 and C226 and C232 forms a disulfide bridge. We tested this by assessing the effect of mutating the C226 and C232 residues to serine on H 2 S‐regulation of SOCE. This was done by transiently transfecting wild‐type or mutant human Orai3 into HEK293 cells stably expressing human STIM1 and measuring SOCE by Ca 2+ imaging. Treatment with the H 2 S donor GYY4137 (500 μM) inhibited the SOCE mediated by STIM1/Orai3. Mutating either C226 or C232 alone had no effect on SOCE under control conditions, however, the ability of GYY4137 to inhibit SOCE was lost in the mutants. A double mutation of both C226 and C232 also had no effect on SOCE under control conditions and GYY4137 again failed to inhibit SOCE. In addition, pre‐incubation with the reductant dithiothreitol (5 mM) completely blocked the inhibitory effect of GYY4137. These data are consistent with a model in which H 2 S inhibits SOCE mediated by STIM1/Orai3 by targeting a disulfide bridge between C226 and C232. Support or Funding Information Rosalind Franklin University of Medicine and Science