We used the patch-clamp technique to study the effect of H(2)O(2) on the apical ROMK-like small-conductance K (SK) channel in the cortical collecting duct (CCD). The addition of H(2)O(2) decreased the activity of the SK channels and the inhibitory effect of H(2)O(2) was larger in the CCD from rats on a K-deficient diet than that from rats on a normal-K or a high-K diet. However, application of H(2)O(2) did not inhibit the SK channels in inside-out patches. This suggests that the H(2)O(2)-mediated inhibition of SK channels was not due to direct oxidation of the SK channel protein. Because a previous study showed that H(2)O(2) stimulated the expression of Src family protein tyrosine kinase (PTK) which inhibited SK channels (3), we explored the role of PTK in mediating the effect of H(2)O(2) on SK channels. The application of H(2)O(2) stimulated the activity of endogenous PTK in M-1 cells and increased tyrosine phosphorylation of ROMK in HEK293 cells transfected with GFP-ROMK1 and c-Src. However, blockade of PTK only attenuated but did not completely abolish the inhibitory effect of H(2)O(2) on SK channels. Since H(2)O(2) has also been demonstrated to activate mitogen-activated protein kinase, P38, and ERK (3), we examined the role of P38 and ERK in mediating the effect of H(2)O(2) on SK channels. Similar to blockade of PTK, suppression of P38 and ERK did not completely abolish the H(2)O(2)-induced inhibition of SK channels. However, combined use of ERK, P38, and PTK inhibitors completely abolished the effect of H(2)O(2) on SK channels. Also, treatment of the CCDs with concanavalin A, an agent which has been shown to inhibit endocytosis (19), abolished the inhibitory effect of H(2)O(2). We conclude that addition of H(2)O(2) inhibited SK channels by stimulating PTK activity, P38, and ERK in the CCD and that H(2)O(2) enhances the internalization of the SK channels.