Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain, and other peripheral tissues. Phosphatidylinositol 4,5-bisphosphate (PIP₂) is a key direct activator of ion channels, including Kir channels. The gasotransmitter carbon monoxide has been shown to regulate Kir channel activity by altering channel-PIP₂ interactions. Here, we tested in two cellular models the effects and mechanism of action of another gasotransmitter, hydrogen sulfide (H₂S), thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide as an exogenous H₂S source and expression of cystathionine γ-lyase, a key enzyme that produces endogenous H₂S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. This effect resulted from changes in channel-gating kinetics rather than in conductance or cell-surface localization. The extent of H₂S regulation depended on the strength of the channel-PIP₂ interactions. H₂S regulation was attenuated when channel-PIP₂ interactions were strengthened and was increased when channel-PIP₂ interactions were weakened by depleting PIP₂ levels. These H₂S effects required specific cytoplasmic cysteine residues in Kir3.2 channels. Mutation of these residues abolished H₂S inhibition, and reintroduction of specific cysteine residues back into the background of the cytoplasmic cysteine-lacking mutant rescued H₂S inhibition. Molecular dynamics simulation experiments provided mechanistic insights into how potential sulfhydration of specific cysteine residues could lead to changes in channel-PIP₂ interactions and channel gating.

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