The Kir4.1 channel is expressed in the brainstem, retina and kidney where it acts on K(+) transportation and pH-dependent membrane potential regulation. Its heteromerization with Kir5.1 leads to K(+) currents with distinct properties such as single-channel conductance, rectification, pH sensitivity and phosphorylation modulation. Here we show that Kir5.1 also enables S-glutathionylation to the heteromeric channel. Expressed in HEK cells, an exposure to the oxidant H(2)O(2) or diamide produced concentration-dependent inhibitions of the whole-cell Kir4.1-Kir5.1 currents. In inside-out patches, currents were inhibited strongly by a combination of diamide/GSH or H(2)O(2)/GSH but not by either alone. The currents were also suppressed by GSSG and the thiol oxidants pyridine disulfides (PDSs), suggesting S-glutathionylation. In contrast, none of the exposures had significant effects on the homomeric Kir4.1 channel. Cys158 in the TM2 helix of Kir5.1 was critical for the S-glutathionylation, which was accessible to intracellular but not extracellular oxidants. Site-directed mutagenesis of this residue (C158A or C158T) abolished the Kir4.1-Kir5.1 current modulation by oxidants, and eliminated almost completely the biochemical interaction of Kir5.1 with GSH. In tandem Kir4.1-Kir5.1 constructs, the channel with a single Cys158 was inhibited to the same degree as the wild-type channel, suggesting that one glutathione moiety is sufficient to block the channel. Consistent with the location of Cys158, GSSG inhibited the channel only when the channel was open, indicating that the channel inhibition was state dependent. The finding that the heteromeric Kir4.1-Kir5.1 channel but not the homomeric Kir4.1 is subject to the S-glutathionylation thus suggests a novel Kir4.1-Kir5.1 channel modulation mechanism that is likely to occur in oxidative stress.