Effect of hydrogen peroxide on ROMK channels in the cortical collecting duct

Abstract

We used the patch-clamp technique to study the effect of H2O2 on the apical ROMK-like small-conductance K (SK) channel in the cortical collecting duct (CCD). The addition of H2O2 decreased the activity of the SK channels and the inhibitory effect of H2O2 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 H2O2 did not inhibit the SK channels in inside-out patches. This suggests that the H2O2-mediated inhibition of SK channels was not due to direct oxidation of the SK channel protein.Because a previous study showed that H2O2 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 H2O2 on SK channels. The application of H2O2 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 H2O2 on SK channels. Since H2O2 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 H2O2 on SK channels. Similar to blockade of PTK, suppression of P38 and ERK did not completely abolish the H2O2-induced inhibition of SK channels. However, combined use of ERK, P38, and PTK inhibitors completely abolished the effect of H2O2 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 H2O2. We conclude that addition of H2O2 inhibited SK channels by stimulating PTK activity, P38, and ERK in the CCD and that H2O2 enhances the internalization of the SK channels.

Role of gp91phox-Containing NADPH Oxidase in Mediating the Effect of K Restriction on ROMK Channels and Renal K Excretion

“…We and others have shown that the K restriction decreased ROMK channel activity and caused the internalization of ROMK channels. 13,26,27 Although the molecular mechanism by which low K suppresses ROMK channel activity and renal K secretion is not completely understood, our previous study suggests that superoxide anions and related products are signaling molecules that mediate the effect of low K intake on ROMK channels and renal K secretion 4,7,28 Furthermore, we show that P38 and ERK MAPK and PTK are involved in mediating the effect of superoxide anions on ROMK channels. 4,7,28 The role of P38 and ERK MAPK in mediating the effect of low K intake and superoxide anions on ROMK channels is supported by several lines of evidence: (1) Application of hydrogen peroxide stimulated the phosphorylation of ERK and P38 MAPK in M-1 cells; (2) inhibition of superoxide anion production with tempol abolished the stimulatory effect of low K intake on the phosphorylation of P38 and ERK; and (3) suppression of P38 and ERK MAPK increased ROMK channel activity and diminished the inhibition of ROMK channels induced by hydrogen peroxide in the CCD.…”

“…13,26,27 Although the molecular mechanism by which low K suppresses ROMK channel activity and renal K secretion is not completely understood, our previous study suggests that superoxide anions and related products are signaling molecules that mediate the effect of low K intake on ROMK channels and renal K secretion 4,7,28 Furthermore, we show that P38 and ERK MAPK and PTK are involved in mediating the effect of superoxide anions on ROMK channels. 4,7,28 The role of P38 and ERK MAPK in mediating the effect of low K intake and superoxide anions on ROMK channels is supported by several lines of evidence: (1) Application of hydrogen peroxide stimulated the phosphorylation of ERK and P38 MAPK in M-1 cells; (2) inhibition of superoxide anion production with tempol abolished the stimulatory effect of low K intake on the phosphorylation of P38 and ERK; and (3) suppression of P38 and ERK MAPK increased ROMK channel activity and diminished the inhibition of ROMK channels induced by hydrogen peroxide in the CCD. 28 The evidence to support the role of PTK in mediating the effect of low K intake and superoxide anions on ROMK channel activity 26 includes the following: (1) Low K intake significantly increased the expression of Src family PTK and suppression of superoxide anions abolished the effect of low K intake on PTK expression, 4 (2) increased PTK activity enhanced the tyrosine phosphorylation of ROMK channels 29 and enhanced the internalization of ROMK channels, 8 and (3) inhibition of PTK attenuated the hydrogen peroxide-induced inhibition of ROMK channels in the CCD.…”

“…4,7,28 The role of P38 and ERK MAPK in mediating the effect of low K intake and superoxide anions on ROMK channels is supported by several lines of evidence: (1) Application of hydrogen peroxide stimulated the phosphorylation of ERK and P38 MAPK in M-1 cells; (2) inhibition of superoxide anion production with tempol abolished the stimulatory effect of low K intake on the phosphorylation of P38 and ERK; and (3) suppression of P38 and ERK MAPK increased ROMK channel activity and diminished the inhibition of ROMK channels induced by hydrogen peroxide in the CCD. 28 The evidence to support the role of PTK in mediating the effect of low K intake and superoxide anions on ROMK channel activity 26 includes the following: (1) Low K intake significantly increased the expression of Src family PTK and suppression of superoxide anions abolished the effect of low K intake on PTK expression, 4 (2) increased PTK activity enhanced the tyrosine phosphorylation of ROMK channels 29 and enhanced the internalization of ROMK channels, 8 and (3) inhibition of PTK attenuated the hydrogen peroxide-induced inhibition of ROMK channels in the CCD. 28 Because low K-induced increases in PTK expression occurs later than that of the phosphorylation of P38 and ERK MAPK, it is possible that the inhibitory effect of low K intake on ROMK channels and renal K secretion is first achieved by activation of MAPK.…”

“…28 The evidence to support the role of PTK in mediating the effect of low K intake and superoxide anions on ROMK channel activity 26 includes the following: (1) Low K intake significantly increased the expression of Src family PTK and suppression of superoxide anions abolished the effect of low K intake on PTK expression, 4 (2) increased PTK activity enhanced the tyrosine phosphorylation of ROMK channels 29 and enhanced the internalization of ROMK channels, 8 and (3) inhibition of PTK attenuated the hydrogen peroxide-induced inhibition of ROMK channels in the CCD. 28 Because low K-induced increases in PTK expression occurs later than that of the phosphorylation of P38 and ERK MAPK, it is possible that the inhibitory effect of low K intake on ROMK channels and renal K secretion is first achieved by activation of MAPK. 7 However, it is safe to conclude that superoxide anions mediate the effect of low K intake on ROMK channels and renal K secretion through MAPK and PTK.…”

Inhibition of ROMK channels by low extracellular K+ and oxidative stress

“…In particular, when the channels are closed by low pH on the cytoplasmic side of the patch, recovery is greatly accelerated by addition of a reducing agent such as DTT (30,46 To test this further, we incubated intact tubules with an oxidizing agent, H 2 O 2 , added to the bath. This compound was previously shown to inhibit ROMK channels in cell-attached patches from the CCD (40). TPNQ-sensitive currents were then measured immediately after formation of whole-cell conditions to assess the response of the intact cell to the oxidant.…”