The effects of oxidizing and cysteine-reactive reagents on the inward rectifier potassium channels Kir2.3 and Kir1.1

The inwardly rectifying potassium channel Kir2.3 possesses extracellular cy steine residues at positions 113, 140 and 145, as well as at position 79 ne ar the outer membrane boundary. Ln this study, we have investigated the rol es of these extracellular cysteine residues in mediating inhibition of the Kir2.3 channel by the cysteine-reactive reagents para-chloromercuribenzenes ulphonate (PCMBS) and thimerosal, and the oxidizing agent hydrogen peroxide (H2O2). We have also compared the effects of these reagents with those on Kir1.1 channels (which do not: possess cysteine residues equivalent to 140 and 79 in Kir2.3 channels). Mutant channels were made in which cysteine res idues were mutated to serine by site-directed mutagenesis. Wild-type or mut ant: cRNA was injected into Xenopus oocytes and voltage-clamp recordings ma de 1-2 days later. Wild-type Kir2.3 currents were significantly inhibited b y PCMBS, thimerosal and H2O2. Currents for mutants Kir2.3 C79S and C140S we re also inhibited by PCMBS, thimerosal and H2O2. These mutations affected t he time course of inhibition by all three reagents. For PCMBS, a slow compo nent of inhibition was absent for the C79S mutation, and a fast component w as absent for C140S. For the double mutation C79S/C140S, PCMBS no longer ha d any effect. For thimerosal, there was a slower time course for C140S, a f aster time course for C79S, and a delayed onset for C79S/C140S. For H2O2, t he main effect was a delayed onset fur the double mutant. The reducing agen t dithiothreitol (DTT) reversed the inhibition by both PCMBS and thimerosal of wild-type and mutant currents, but not the inhibition due to H2O2 . Fin ally, wildtype Kir1.1 currents were not significantly inhibited by the appl ication of either PCMBS or thimerosal, while H2O2, produced small inhibitio n. The results taken together indicate that inhibition by the cysteine-reac tive reagent PCMBS is mediated through cysteine residues 79 and 140 in Kir2 .3 channels,with C79 mediating a slow component of inhibition and C140 a fa ster component, and that both residues are extracellularly exposed. The dat a indicate that these two cysteine residues are also main sites for inhibit ion by thimerosal and H2O2 but unlike for PCMBS, an additional non-extracel lular inhibitory site(s) must also be involved.