K restriction inhibits protein phosphatase 2B (PP2B) and suppression of PP2B decreases ROMK channel activity in the CCD

Abstract

We used Western blot analysis to examine the effect of dietary K intake on the expression of serine/threonine protein phosphatase in the kidney. K restriction significantly decreased the expression of catalytic subunit of protein phosphatase (PP)2B but increased the expression of PP2B regulatory subunit in both rat and mouse kidney. However, K depletion did not affect the expression of PP1 and PP2A. Treatment of M-1 cells, mouse cortical collecting duct (CCD) cells, or 293T cells with glucose oxidase (GO), which generates superoxide anions through glucose metabolism, mimicked the effect of K restriction on PP2B expression and significantly decreased expression of PP2B catalytic subunits. However, GO treatment increased expression of regulatory subunit of PP2B and had no effect on expression of PP1, PP2A, and protein tyrosine phosphatase 1D. Moreover, deletion of gp91-containing NADPH oxidase abolished the effect of K depletion on PP2B. Thus superoxide anions or related products may mediate the inhibitory effect of K restriction on the expression of PP2B catalytic subunit. We also used patch-clamp technique to study the effect of inhibiting PP2B on renal outer medullary K (ROMK) channels in the CCD. Application of cyclosporin A or FK506, inhibitors of PP2B, significantly decreased ROMK channels, and the effect of PP2B inhibitors was abolished by blocking p38 mitogen-activated protein kinase (MAPK) and ERK. Furthermore, Western blot demonstrated that inhibition of PP2B with cyclosporin A or small interfering RNA increased the phosphorylation of ERK and p38 MAPK. We conclude that K restriction suppresses the expression of PP2B catalytic subunits and that inhibition of PP2B decreases ROMK channel activity through stimulation of MAPK in the CCD.

Fig. 1

Effect of dietary K intake on the expression of protein phosphatase (PP)1 (n = 3 rats; A), PP2A (n = 3 rats; B), and PP2B catalytic subunit (PP2B-cat, 5 rats; C). Top: corresponding Western blots. Bottom: summary of data. Data are normalized according to corresponding control. Arrow indicates the position of each corresponding phosphatase that is quantified and presented in the bar graph. PP2B-cat protein purified from mouse brain (input) was used as a positive control. NK, normal K diet; KD, K-deficient diet; OM, outer medulla. *Significant difference (P < 0.05).

Fig. 2

Effect of glucose oxidase (GO) treatment on the expression of PP2B-cat in M-1 cells (A) and in 293T cells (B). Top: corresponding Western blots. Bottom: summary of data. Data are normalized according to corresponding control. Cells were treated with GO (1 or 5 U/ml) for 15 min, followed by homogenization. Arrow indicates the position of PP2B-cat. *Significant difference (P < 0.05).

Fig. 3

Effect of GO treatment (1 U/ml) on the expression of PP2A (left), PP1 (center), and protein tyrosine phosphatase (PTP)1D (right) in 293 T cells. Cells were treated with GO for 15 or 30 min. Arrow indicates the position of each corresponding phosphatase.

Fig. 4

A: effect of K restriction on the expression of PP2B regulatory subunit (PP2B-R) in rat. HK, High K diet. B: effect of K restriction on the expression of PP2B-cat in wild-type (WT) mice or in gp91^phox(-/-) (KO) mice. C: effect of K restriction on the expression of PP2B-R in WT and gp91^phox(-/-) mice. Arrow indicates the position of PP2B-cat or PP2B-R. Right: summary of results for each corresponding experiment. *Significant difference (P < 0.05).

Fig. 5

Effect of FK506 (5 μM) on the renal outer medullary K (ROMK) channels in the cortical collecting duct (CCD). A: top trace shows the time course of the experiments, and 2 parts of the recording indicated with bars and numbers are extended to show the fast time resolution. The channel close level is indicated by a dotted line and C. The gap in the top trace is 300 s. The pipette hold potential was 0 mV. B: effect of FK506/cyclosporin A on ROMK channels in CCDs, which were treated with or without 5 μM SB-202190 (SB) + 50 μM PD-098059 (PD). Experiments were performed in cell-attached patches. *Significant difference (P < 0.05).

Fig. 6

Top: Western blot showing the effect of cyclosporin A (5 μM) treatment (60 min) on the phosphorylation of ERK and p38 detected with phospho-ERK and phospho-p38 antibodies. Control cells were treated with vehicles for 0 or 60 min. Bottom: effect of cyclosporin A (5 μM) on the phosphorylation of p38 and ERK. Arrow indicates the position of p38 and ERK, which is quantified and presented in the corresponding bar graph. The phosphorylation level of ERK and p38 at the 60-min band was used as control value. *Significant difference (P < 0.05).

Fig. 7

Effect of PP2B small interfering (si)RNA on the expression of PP2B-cat and actin (A) and the phosphorylation of ERK and p38 in 293T cells that were transfected with siRNA or mock-transfected for 24 h (B). Bottom: summary of results of the experiments. *Significant difference (P < 0.05).

Fig. 8

Scheme illustrating the possible role of PP2B in mediating the effect of low K intake on apical small-conductance K (SK) and big conductance K (BK) channels in the CCD. A dotted arrow indicates a diminished effect. ING4, inhibitor of growth 4; PTK, protein tyrosine kinases.