ENaC activity is increased in isolated, split-open cortical collecting ducts from protein kinase Cα knockout mice

Abstract

The epithelial Na channel (ENaC) is negatively regulated by protein kinase C (PKC) as shown using PKC activators in a cell culture model. To determine whether PKC␣ influences ENaC activity in vivo, we examined the regulation of ENaC in renal tubules from PKC␣ Ϫ/Ϫ mice. Cortical collecting ducts were dissected and split open, and the exposed principal cells were subjected to cell-attached patch clamp. In the absence of PKC␣, the open probability (P o) of ENaC was increased three-fold vs. wild-type SV129 mice (0.52 Ϯ 0.04 vs. 0.17 Ϯ 0.02). The number of channels per patch was also increased. Using confocal microscopy, we observed an increase in membrane localization of ␣-, ␤-, and ␥-subunits of ENaC in principal cells in the cortical collecting ducts of PKC␣ Ϫ/Ϫ mice compared with wild-type mice. To confirm this increase, one kidney from each animal was perfused with biotin, and membrane protein was pulled down with streptavidin. The nonbiotinylated kidney was used to assess total protein. While total ENaC protein did not change in PKC␣ Ϫ/Ϫ mice, membrane localization of all the ENaC subunits was increased. The increase in membrane ENaC could be explained by the observation that ERK1/2 phosphorylation was decreased in the knockout mice. These results imply a reduction in ENaC membrane accumulation and P o by PKC␣ in vivo. The PKC-mediated increase in ENaC activity was associated with an increase in blood pressure in knockout mice fed a high-salt diet.protein kinase C␣; ENaC; renal tubules; single channels; knockout mice; hypertension EPITHELIAL NA CHANNELS (ENaC) are sodium-permeable ion channels located in the apical membrane of polarized epithelial cells primarily in the distal nephron, lung, and distal colon. In the distal nephron, ENaC activity is the rate-limiting step for Na ϩ reabsorption (16, 34); therefore, ENaC activity is critical for the physiological maintenance of systemic Na ϩ homeostasis and long-term control of blood pressure. Because of its central role in responding to changes in Na ϩ uptake, ENaC activity is tightly regulated; dysregulation of this channel has been linked to abnormal blood pressure in several genetic disorders including Liddle's syndrome (18, 37) and pseudohypoaldosteronism type 1 (9, 33, 41).ENaC can be regulated either by altering the amount of time the channel spends open (open probability or P o ) or by altering the density of functional channels (N) in the apical membrane of distal nephron epithelial cells. One signaling molecule that appears to alter ENaC activity is protein kinase C (PKC). Activation of PKC with phorbol esters reduces ENaC activity in the apical membrane of A6 cells, an amphibian renal cell line, and in rat principal cells (15). In contrast to the inhibitory effect on ENaC due to activating PKC, pharmacologically inhibiting PKC increases ENaC P o (23, 49). A6 cells, on which many of the experiments described above were performed, contain several different PKC isoforms; so that it is difficult to determine which isoform is responsible for the changes in E...

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

“…However, to determine the levels of ROMK1 (whether as the higher or lower molecular mass form) in the apical membranes of renal tubules, we performed in situ biotinylation experiments, which we have previously reported. 26 Interestingly, much higher molecular mass ROMK1 than lower was detected in the apical membranes of renal tubules ( Figure 1C); 100 mM DTT significantly decreased the amount of higher molecular mass ROMK1 in the biotinylated apical membranes of renal tubules (Figure 1, D and E). As shown in Supplemental Figure 1, the single band detected by an ROMK antibody from Alomone Laboratories was significantly reduced in mpkCCDc14 cells transiently transfected with siRNA against ROMK1 but was not altered by control siRNA; 100 mM DTT caused disappearance of the higher molecular mass band and induced appearance of another band with a molecular mass ,50 kD.…”

“…In situ biotinylation of the apical membranes of renal tubules was performed like we previously described. 26 Either the intact apical membrane of control mpkCCD c14 cells containing both microvilli and planar regions or the apical membrane without microvilli (eliminated with MbCD as depicted in Figure 5C) was biotinylated and collected. These membranes and lysates from MbCD-released vesicles containing microvilli were loaded and electrophoresed on 10% SDS-PAGE gels for 60-90 minutes.…”

Pendrin gene ablation alters ENaC subcellular distribution and open probability

“…A similar mechanism may contribute to pendrin-dependent changes in ENaC open probability (P o ) or subcellular distribution, since channel activity (or NP o ) in cultured A6 Xenopus cells is virtually eliminated when the HCO 3 Ϫ concentration in culture media is reduced from 25 to 5 mM (25). However, the effect of apical HCO 3 Ϫ concentration (or pH) on ENaC open probability and subcellular distribution remains to be determined.…”

“…epithelial sodium channel; pendrin PENDRIN, encoded by Slc26a4, is an aldosterone-sensitive, electroneutral, Na ϩ -independent Cl Ϫ /HCO 3 Ϫ exchanger that is expressed in the apical regions of renal intercalated cells (1,14,28,30,31,36,39,40). With increased circulating aldosterone, increased pendrin-mediated Cl Ϫ absorption and HCO 3 Ϫ secretion are observed, which secondarily stimulates Na ϩ absorption, thereby contributing to the pressor response to this steroid hormone (36).…”

ENaC inhibition stimulates HCl secretion in the mouse cortical collecting duct. I. Stilbene-sensitive Cl−secretion

“…Electrophysiological measurements were made in split open-mouse CCDs, as previously described (2). Aldosterone-treated collecting duct-specific ENaC-null mice (treatment 2) were euthanized by an overdose of pentobarbital followed by cervical dislocation, and the kidneys were immediately removed.…”