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. potassium depletion; p38 mitogen-activated protein kinase; extracellular signal-regulated kinase 1/2; potassium secretion THE KIDNEY PLAYS A KEY ROLE in maintaining plasma K level in a normal range by secretion of K to match the dietary intake (16). Renal K secretion mainly takes place in the late distal tubule, the connecting tubule, and the cortical collecting duct (CCD) by a two-step mechanism (16, 35): K enters the cell through Na-K-ATPase at the basolateral membrane and leaves the cell through apical K channels. Although a Ca 2ϩ -activated big-conductance K (BK) channel has been shown to be involved in K secretion during high K intake or increasing tubule flow rate (6,36,43), renal outer medullary K (ROMK)-like small-conductance K (SK) channels play a major role in K secretion under normal K intake.Dietary K intake is an important factor regulating renal K secretion (13,19,33): increasing dietary K intake stimulates whereas decreasing K intake suppresses renal K secretion. However, the signaling pathway by which low K intake suppresses renal K secretion is not completely understood. We (3, 4) and others (24) have demonstrated that protein tyrosine kinases (PTK) and serine/threonine protein kinases are involved in mediating the effect of K restriction on SK channels. In ...

NAD(P)H oxidase and renal epithelial ion transport

“…The reduction in ROMK-like SK channel activity following inhibition of PP2B-cat could be reversed by coincubation with p38 and ERK inhibitors, suggesting MAPKs involvement in ROMK-like SK inactivation (51). As inhibition of PP2B-cat increases the phosphorylation of p38 and ERK, the authors concluded that ROS can inhibit the ROMK-like SK channel by reducing PP2B-cat activity (51). This would increase p38 and ERK phosphorylation, activating them, which would increase ROMK-like SK channel phosphorylation, inhibiting it.…”

“…Exogenous O 2 Ϫ or H 2 O 2 was found to reduce the expression of PP2B-cat (catalytic subunit) in M1 cells (51). Further, inhibition of PP2B-cat using cyclosporine A or FK-506 reduced apical ROMK-like SK channel activity (51).…”

“…Further, inhibition of PP2B-cat using cyclosporine A or FK-506 reduced apical ROMK-like SK channel activity (51). The reduction in ROMK-like SK channel activity following inhibition of PP2B-cat could be reversed by coincubation with p38 and ERK inhibitors, suggesting MAPKs involvement in ROMK-like SK inactivation (51). As inhibition of PP2B-cat increases the phosphorylation of p38 and ERK, the authors concluded that ROS can inhibit the ROMK-like SK channel by reducing PP2B-cat activity (51).…”

“…Exogenous O 2 Ϫ or H 2 O 2 was found to reduce the expression of PP2B-cat (catalytic subunit) in M1 cells (51). Further, inhibition of PP2B-cat using cyclosporine A or FK-506 reduced apical ROMK-like SK channel activity (51). The reduction in ROMK-like SK channel activity following inhibition of PP2B-cat could be reversed by coincubation with p38 and ERK inhibitors, suggesting MAPKs involvement in ROMK-like SK inactivation (51).…”

Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

“…9 This channel may be responsible for drug-induced hyperkalemia, because cyclosporin A (CsA), an immunosuppressant drug that causes hyperkalemia, inhibits the channel. 10,11 However, the mechanism by which CsA inhibits ROMK1 channels remains unclear. Our recent studies suggest that CsA stimulates the epithelial sodium channel (ENaC) through elevation of intracellular cholesterol.…”