Mitogen-activated protein kinases inhibit the ROMK (Kir 1.1)-like small conductance K channels in the cortical collecting duct

Abstract

It was demonstrated previously that low dietary potassium (K) intake stimulates Src family protein tyrosine kinase (PTK) expression via a superoxide-dependent signaling. This study explored the role of mitogen-activated protein kinase (MAPK) in mediating the effect of superoxide anions on PTK expression and ROMK (Kir 1.1) channel activity. Western blot analysis demonstrated that low K intake significantly increased the phosphorylation of P38 MAPK (P38) and extracellular signal-regulated kinase (ERK) but had no effect on phosphorylation of c-JUN N-terminus kinase in renal cortex and outer medulla. The stimulatory effect of low K intake on P38 and ERK was abolished by treatment of rats with tempol. The possibility that increases in superoxide and related products that are induced by low K intake were responsible for stimulating phosphorylation of P38 and ERK also was supported by the finding that application of H(2)O(2) increased the phosphorylation of ERK and P38 in the cultured mouse collecting duct cells. Simultaneous blocking of ERK and P38 completely abolished the effect of H(2)O(2) on c-Src expression in mouse collecting duct cells. For determination of the role of P38 and ERK in the regulation of ROMK-like small-conductance K (SK) channels, the patch-clamp technique was used to study the effect of inhibiting P38 and ERK on SK channels in the cortical collecting duct from rats that were on a control K diet (1.1%) and on a K-deficient diet for 1 d. Inhibition of ERK, c-JUN N-terminus kinase, or P38 alone had no effect on SK channels. In contrast, simultaneous inhibition of P38 and ERK significantly increased channel activity. The effect of inhibiting MAPK on SK channels was not affected in the presence of herbimycin A, a PTK inhibitor, and was larger in rats that were on a K-deficient diet than in rats that were on a normal-K diet. However, the stimulatory effect of inhibiting ERK and P38 on SK was absent in the cortical collecting duct that was treated with colchicine. It is concluded that low K intake-induced increases in superoxide levels are responsible for stimulation of P38 and ERK and that MAPK inhibit the SK channels by stimulating PTK expression and via a PTK-independent mechanism.

Figure 1

Effect of potassium (K) depletion on the phosphorylation of P38 mitogen-activated protein kinase (P38 MAPK; A), extracellular signal–regulated kinase (ERK; B), and c-JUN N-terminus kinase (JNK; C) in rats that received tempol treatment or vehicle. (Top) Phosphorylated P38, ERK, and JNK, respectively. (Middle) Corresponding total amount of P38, ERK, and JNK. Data are summarized as a bar graph at the bottom of each panel. Changes of the band intensity are determined by calculation of the ratio between phosphorylation band intensity (Exp)/control value times the ratio between total protein intensity (normal)/Exp. *P < 0.01, control versus experimental group.

Figure 2

Effect of H₂O₂ on the phosphorylation of P38 MAPK (A), ERK (B), and JNK (C) in mouse collecting duct (M-1) cells. (Top and middle) Phosphorylated MAPK and the total MAPK, respectively. Data are summarized as a bar graph at the bottom of each panel. *P < 0.01, control versus experimental group.

Figure 3

(A) Effect of H₂O₂ on the expression of c-Src in M-1 cells in the absence of MAPK inhibitors and in the presence of PD098059 (50 µM), SP600125 (10 µM), SB202190 (5 µM), PD+SB, or three inhibitors. Data are summarized and presented as bar graph at the bottom of the figure. **P < 0.001 and *P < 0.01, control versus experimental group. IP, immunoprecipitation; IB, immunoblot. (B) A Western blot showing the effect of H₂O₂ on the expression of c-Src in M-1 cells in the presence of SP600125+SB202190, SP600125+PD098059, and SB202190+PD098059.

Figure 4

A channel recording showing that simultaneous inhibition of ERK with PD098059 (PD) and P38 MAPK with SB202190 (SB) activates the small-conductance K (SK) channels in the cortical collecting duct (CCD) from a rat that was on a normal K diet. The experiments were performed in cell-attached patches, and the holding potential was 30 mV. The top trace is the time course of the experiment, whereas three parts of trace, indicated by numbers, are extended to show the fast time resolution. The channel close level is indicated by “C.” The arrow indicates the addition of MAPK inhibitors that were present throughout the experiments. Also, the electric noise after addition of PD+SB was artifact.

Figure 5

Effect of PD098059+SB202190 on the SK channels in the presence and absence of herbimycin A in the CCD from rats that were on a normal K diet. The experiments were performed in cell-attached patches. Herbimycin A (1 µM) was used to treated the CCD for 15 min and present throughout the experiment. *P < 0.05 and **P < 0.01 versus control.

Figure 6

A channel recording showing that simultaneous inhibition of ERK and P38 MAPK activates the SK channels in the presence of herbimycin A. The experiments were performed in cell-attached patches, and the holding potential was 30 mV. The top trace is the time course of the experiment, whereas three parts of trace, indicated by numbers, are extended to show the fast time resolution. The channel close level is indicated by “C,” and the gap of the top trace is 180 s. The arrow indicates the addition of MAPK inhibitors, which were present throughout the experiments.

Figure 7

(A) A bar graph shows the effect of K restriction for 1 d on superoxide levels (n = 3). *Significant difference. (B) Western blots demonstrate the effect of 24-h K restriction on the phosphorylation of P38 and ERK and the expression of c-Src. (C) Effect of K restriction for 24 h on the SK channel activity in the presence or absence of inhibitors of MAPK (PD098059+ SB202190). *Significant different between control and K-deficient (KD) group; ^#significant difference between KD and KD+MAPK inhibitor groups. The experimental number is indicated under each bar.

Figure 8

A cell model illustrating the role of MAPK in mediating the effect of low K intake on ROMK (Kir 1.1) channel activity in the CCD.