Oxygen regulation of the epithelial Na channel in the collecting duct

The PO(2) within the kidney changes dramatically from cortex to medulla. The present experiments examined the effect of changing PO(2) on epithelial Na channel (ENaC)-mediated Na transport in the collecting duct using the mpkCCD-c14 cell line. Decreasing ambient O(2) concentration from 20 to 8% decreased ENaC activity by 40%; increasing O(2) content to 40% increased ENaC activity by 50%. The O(2) effect required several hours to develop and was not mimicked by the acid pH that developed in monolayers incubated in low-O(2) medium. Corticosteroids increased ENaC activity at each O(2) concentration; there was no interaction. The pathways for O(2) and steroid regulation of ENaC are different since O(2) did not substantially affect Sgk1, α-ENaC, Gilz, or Usp2-45 mRNA levels, genes involved in steroid-mediated ENaC regulation. The regulation of ENaC activity by these levels of O(2) appears not to be mediated by changes in hypoxia-inducible factor-1α or -2α activity or a change in AMP kinase activity. Changes in O(2) concentration had minimal effect on α- or γ-ENaC mRNA and protein levels; there were moderate effects on β-ENaC levels. However, 40% O(2) induced substantially greater total β- and γ-ENaC on the apical surface compared with 8% O(2); both subunits demonstrated a greater increase in the mature forms. The α-ENaC subunit was difficult to detect on the apical surface, perhaps because our antibodies do not recognize the major mature form. These results identify a mechanism of ENaC regulation that may be important in different regions of the kidney and in responses to changes in dietary NaCl.     

N-methyl-D-aspartate receptor subunit NR3a expression and function in principal cells of the collecting duct

N-methyl-D-aspartate receptors (NMDARs) are Ca(2+)-permeable, ligand-gated, nonselective cation channels that function as neuronal synaptic receptors but which are also expressed in multiple peripheral tissues. Here, we show for the first time that NMDAR subunits NR3a and NR3b are highly expressed in the neonatal kidney and that there is continued expression of NR3a in the renal medulla and papilla of the adult mouse. NR3a was also expressed in mIMCD-3 cells, where it was found that hypoxia and hypertonicity upregulated NR3a expression. Using short-hairpin (sh) RNA-based knockdown, a stable inner medullary collecting duct (IMCD) cell line was established that had ～80% decrease in NR3a. Knockdown cells exhibited an increased basal intracellular calcium concentration, reduced cell proliferation, and increased cell death. In addition, NR3a knockdown cells exhibited reduced water transport in response to the addition of vasopressin, suggesting an alteration in aquaporin-2 (AQP2) expression/function. Consistent with this notion, we demonstrate decreased surface expression of glycosylated AQP2 in IMCD cells transfected with NR3a shRNA. To determine whether this also occurred in vivo, we compared AQP2 levels in wild-type vs. in NR3a(-/-) mice. Total AQP2 protein levels in the outer and inner medulla were significantly reduced in knockout mice compared with control mice. Finally, NR3a(-/-) mice showed a significant delay in their ability to increase urine osmolality during water restriction. Thus NR3a may play a renoprotective role in collecting duct cells. Therefore, under conditions that are associated with high vasopressin levels, NR3a, by maintaining low intracellular calcium levels, protects the function of the principal cells to reabsorb water and thereby increase medullary osmolality.     

Plasmin activates epithelial Na+ channels by cleaving the gamma subunit

Proteolytic processing of epithelial sodium channel (ENaC) subunits occurs as channels mature within the biosynthetic pathway. The proteolytic processing events of the alpha and gamma subunits are associated with channel activation. Furin cleaves the alpha subunit ectodomain at two sites, releasing an inhibitory tract and activating the channel. However, furin cleaves the gamma subunit ectodomain only once. A second distal cleavage in the gamma subunit induced by other proteases, such as prostasin and elastase, is required to release a second inhibitory tract and further activate the channel. We found that the serine protease plasmin activates ENaC in association with inducing cleavage of the gamma subunit at gammaLys194, a site distal to the furin site. A gammaK194A mutant prevented both plasmin-dependent activation of ENaC and plasmin-dependent production of a unique 70-kDa carboxyl-terminal gamma subunit cleavage fragment. Plasmin-dependent cleavage and activation of ENaC may have a role in extracellular volume expansion in human disorders associated with proteinuria, as filtered plasminogen may be processed by urokinase, released from renal tubular epithelium, to generate active plasmin.     

A new subunit of the epithelial Na+ channel identifies regions involved in Na+ self-inhibition

The epithelial Na+ channel (ENaC) is the apical entry pathway for Na+ in many Na+-reabsorbing epithelia. ENaC is a heterotetrameric protein composed of homologous alpha, beta, and gamma subunits. Mutations in ENaC cause severe hypertension or salt wasting in humans; and consequently, ENaC activity is tightly controlled. According to the concept of Na+ self-inhibition, the extracellular Na+ ion itself can reduce ENaC activity. The molecular basis for Na+ self-inhibition is unknown. Here, we describe cloning of a new ENaC subunit from Xenopus laevis (epsilonxENaC). epsilonxENaC can replace alphaxENaC and formed functional, highly selective, amiloride-sensitive Na+ channels when coexpressed with betaxENaC and gammaxENaC. Channels containing epsilonxENaC showed strong inhibition by extracellular Na+. This Na+ self-inhibition was significantly slower than for alphaxENaC-containing channels. Using site-directed mutagenesis, we show that the proximal part of the large extracellular domain controls the speed of self-inhibition. This suggests that this region is involved in conformational changes during Na+ self-inhibition.     

Paracrine regulation of the epithelial Na+ channel in the mammalian collecting duct by purinergic P2Y2 receptor tone

Growing evidence implicates a key role for extracellular nucleotides in cellular regulation, including of ion channels and renal function, but the mechanisms for such actions are inadequately defined. We investigated purinergic regulation of the epithelial Na+ channel (ENaC) in mammalian collecting duct. We find that ATP decreases ENaC activity in both mouse and rat collecting duct principal cells. ATP and other nucleotides, including UTP, decrease ENaC activity via apical P2Y2 receptors. ENaC in collecting ducts isolated from mice lacking this receptor have blunted responses to ATP. P2Y2 couples to ENaC via PLC; direct activation of PLC mimics ATP action. Tonic regulation of ENaC in the collecting duct occurs via locally released ATP; scavenging endogenous ATP and inhibiting P2 receptors, in the absence of other stimuli, rapidly increases ENaC activity. Moreover, ENaC has greater resting activity in collecting ducts from P2Y2-/- mice. Loss of collecting duct P2Y2 receptors in the knock-out mouse is the primary defect leading to increased ENaC activity based on the ability of direct PLC stimulation to decrease ENaC activity in collecting ducts from P2Y2-/- mice in a manner similar to ATP in collecting ducts from wild-type mice. These findings demonstrate that locally released ATP acts in an autocrine/paracrine manner to tonically regulate ENaC in mammalian collecting duct. Loss of this intrinsic regulation leads to ENaC hyperactivity and contributes to hypertension that occurs in P2Y2 receptor-/- mice. P2Y2 receptor activation by nucleotides thus provides physiologically important regulation of ENaC and electrolyte handling in mammalian kidney.     

Determination of epithelial Na+ channel subunit stoichiometry from single-channel conductances

The epithelial Na(+) channel (ENaC) is a multimeric membrane protein consisting of three subunits, alpha, beta, and gamma. The total number of subunits per functional channel complex has been described variously to follow either a tetrameric arrangement of 2alpha:1beta:1gamma or a higher-ordered stoichiometry of 3alpha:3beta:3gamma. Therefore, while it is clear that all three ENaC subunits are required for full channel activity, the number of the subunits required remains controversial. We used a new approach, based on single-channel measurements in Xenopus oocytes to address this issue. Individual mutations that alter single-channel conductance were made in pore-lining residues of ENaC alpha, beta, or gamma subunits. Recordings from patches in oocytes expressing a single species, wild type or mutant, of alpha, beta, and gamma showed a well-defined current transition amplitude with a single Gaussian distribution. When cRNAs for all three wild-type subunits were mixed with an equimolar amount of a mutant alpha-subunit (either S589D or S592T), amplitudes corresponding to pure wild-type or mutant conductances could be observed in the same patch, along with a third intermediate amplitude most likely arising from channels with at least one wild-type and at least 1 mutant alpha-subunit. However, intermediate or hybrid conductances were not observed with coexpression of wild-type and mutant betaG529A or gammaG534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2alpha, 1beta, and 1gamma come together around central pore.     

Modulation of Na+ transport and epithelial sodium channel expression by protein kinase C in rat alveolar epithelial cells

Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na+ transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased alpha, beta, and gammaENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of alphaENaC expression. The PKC inhibitors bisindolylmaleimide at 2 micromol/L and G?6976 at 2 micromol/L diminished the PMA-induced suppression of alphaENaC expression, while rottlerin at 1 micromol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na+-K+-ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na+ entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca2+-dependent PKC is potentially involved in this response.     

Differential effects of protein kinase C on the levels of epithelial Na+ channel subunit proteins

Regulation of epithelial Na(+) channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit levels, differentially decreasing the levels of both beta and gamma, but not alphaENaC. Temporal regulation of beta and gammaENaC by PKC differed; gammaENaC decreased with a time constant of 3.7 +/- 1.0 h, whereas betaENaC decreased in 13.9 +/- 3. 0 h. Activation of PKC also resulted in a decrease in trans-epithelial Na(+) reabsorption for up to 48 h. PMA activation of PKC resulted in negative feedback inhibition of PKC protein levels beginning within 4 h. Both beta and gammaENaC levels, as well as transport tended toward pretreatment values after 48 h of PMA treatment. PKC inhibitors attenuated the effects of PMA on ENaC subunit levels and Na(+) transport. These results directly show for the first time that PKC differentially regulates ENaC subunit levels by decreasing the levels of beta and gamma but not alphaENaC protein. These results imply a PKC-dependent, long term decrease in Na(+) reabsorption.     

Differentiation of epithelial Na+ channel function. An in vitro model

Confluent monolayers of epithelial cells grown on nonporous support form fluid-filled hemicysts called domes, which reflect active ion transport across the epithelium. Clara-like H441 lung adenocarcinoma cells grown on glass supports and exposed to 50 nM dexamethasone developed domes in a time-dependent fashion. Uplifting of small groups of cells occurred within 6-12 h, well formed domes appeared between 24 and 48 h, and after 7 days, individual domes started to merge. Cells inside of domes compared with those outside domes, or with monolayers not exposed to dexamethasone, differed by higher surfactant production, an increased cytokeratin expression, and the localization of claudin-4 proteins to the plasma membrane. In patch clamp studies, amiloride-blockable sodium currents were detected exclusively in cells inside domes, whereas in cells outside of domes, sodium crossed the membrane through La3+-sensitive nonspecific cation channels. Cells grown on permeable support without dexamethasone expressed amiloride-sensitive currents only after tight electrical coupling was achieved (transepithelial electrical resistance (R(t)) > 1 kilohm). In real-time quantitative PCR experiments, the addition of dexamethasone increased the content of claudin-4, occludin, and Na+ channel gamma-subunit (gamma-ENaC) mRNAs by 1.34-, 1.32-, and 1.80-fold, respectively, after 1 h and was followed by an increase at 6 h in the content of mRNA of alpha- and beta-ENaC and of alpha1- and beta1-Na,K-ATPase. In the absence of dexamethasone, neither change in gene expression nor cell uplifting was observed. Our data suggest that during epithelial differentiation, coordinated expression of tight junction proteins precedes the development of vectorial transport of sodium, which in turn leads to the fluid accumulation in basolateral spaces that is responsible for dome formation.     

Intracellular Na+ controls cell surface expression of Na,K-ATPase via a cAMP-independent PKA pathway in mammalian kidney collecting duct cells

In the mammalian kidney the fine control of Na+ reabsorption takes place in collecting duct principal cells where basolateral Na,K-ATPase provides the driving force for vectorial Na+ transport. In the cortical collecting duct (CCD), a rise in intracellular Na+ concentration ([Na+]i) was shown to increase Na,K-ATPase activity and the number of ouabain binding sites, but the mechanism responsible for this event has not yet been elucidated. A rise in [Na+]i caused by incubation with the Na+ ionophore nystatin, increased Na,K-ATPase activity and cell surface expression to the same extent in isolated rat CCD. In cultured mouse mpkCCDcl4 collecting duct cells, increasing [Na+]i either by cell membrane permeabilization with amphotericin B or nystatin, or by incubating cells in a K(+)-free medium, also increased Na,K-ATPase cell surface expression. The [Na+]i-dependent increase in Na,K-ATPase cell-surface expression was prevented by PKA inhibitors H89 and PKI. Moreover, the effects of [Na+]i and cAMP were not additive. However, [Na+]i-dependent activation of PKA was not associated with an increase in cellular cAMP but was prevented by inhibiting the proteasome. These findings suggest that Na,K-ATPase may be recruited to the cell membrane following an increase in [Na+]i through cAMP-independent PKA activation that is itself dependent on proteasomal activity.     

WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in sodium transport in kidney and other epithelia, and in regulating blood pressure. The channel is composed of three subunits (alphabetagamma) each containing two proline-rich sequences (P1 and P2) at its C-terminus. The P2 regions in human beta and gammaENaC, identical to the rat betagammarENaC, were recently shown to be deleted in patients with Liddle's syndrome (a hereditary form of hypertension), leading to hyperactivation of the channel. Using a yeast two-hybrid screen, we have now identified the rat homologue of Nedd4 (rNedd4) as the binding partner for the P2 regions of beta and gammarENaC. rNedd4 contains a Ca2+ lipid binding (CaLB or C2) domain, three WW domains and a ubiquitin ligase (Hect) domain. Our yeast two-hybrid and in vitro binding studies revealed that the rNedd4-WW domains mediate this association by binding to the P2 regions, which include the PY motifs (XPPXY) of either betarENaC (PPPNY) or gammarENaC (PPPRY). SH3 domains were unable to bind these sequences. Moreover, mutations to Ala of Pro616 or Tyr618 within the betarENaC P2 sequence (to PPANY or PPPNA, respectively), recently described in Liddle's patients, led to abrogation of rNedd4-WW binding. Nedd4-WW domains also bound to the proline-rich C-terminus (containing the sequence PPPAY) of alpharENaC, and endogenous Nedd4 co-immunoprecipitated with alpharENaC expressed in MDCK cells. These results demonstrate that the WW domains of rNedd4 bind to the PY motifs deleted from beta or gammaENaC in Liddle's syndrome patients, and suggest that Nedd4 may be a regulator (suppressor) of the epithelial Na+ channel.     

Regulation of cAMP-sensitive colonic epithelial Na+ channel in oocyte expression system

In amphibian epithelia and in cortical collecting duct the antidiuretic peptide arginine-vasopressin (AVP) stimulates activity of epithelial Na+ channels (ENaCs). Generally, the AVP action upon Na+ (re)absorption is believed to be a cAMP/protein-kinase-A mediated mechanism. In the Xenopus oocyte expression system, however, a clear stimulation of ENaC activity by cAMP could not be reproduced with channel subunits cloned from A6 cells or rat colon. We have recently shown that membrane-permeant 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) stimulates activity of a hybrid ENaC in Xenopus oocytes, that consists of an alpha-subunit cloned from guinea-pig colon and the beta- and gamma-subunit originating from rat colon (gpalpharbetagammaENaC). In the present study, we have further investigated the mechanisms by which cpt-cAMP upregulates gpalpharbetagammaENaC activity. Interestingly, we found AVP to stimulate the gpalpharbetagammaENaC in oocytes. Also, treatment with GTP-gamma-S largely activated this channel. In contrast, as a conflicting result, forskolin had no stimulatory effect on the cAMP-sensitive gpalpharbetagammaENaC. Experiments with Brefeldin A (BFA) or nocodazole suggested that only a minor part of cpt-cAMP-induced activation is probably due to an additional translocation of channel proteins into the oocyte membrane. In conclusion, the stimulatory effect of synthetic cpt-cAMP does not seem to be exclusively provided by classical cAMP/PKA-associated transduction mechanisms, i.e., as in A6 cells.     

Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel

The epithelial Na+ channel (alphabetagammaENaC) regulates salt and fluid homeostasis and blood pressure. Each ENaC subunit contains a PY motif (PPXY) that binds to the WW domains of Nedd4, a Hect family ubiquitin ligase containing 3-4 WW domains and usually a C2 domain. It has been proposed that Nedd4-2, but not Nedd4-1, isoforms can bind to and suppress ENaC activity. Here we challenge this notion and show that, instead, the presence of a unique WW domain (WW3*) in either Nedd4-2 or Nedd4-1 determines high affinity interactions and the ability to suppress ENaC. WW3* from either Nedd4-2 or Nedd4-1 binds ENaC-PY motifs equally well (e.g. Kd approximately 10 microm for alpha- or betaENaC, 3-6-fold higher affinity than WW4), as determined by intrinsic tryptophan fluorescence. Moreover, dNedd4-1, which naturally contains a WW3* instead of WW2, is able to suppress ENaC function equally well as Nedd4-2. Homology models of the WW3*.betaENaC-PY complex revealed that a Pro and Ala conserved in all WW3*, but not other Nedd4-WW domains, help form the binding pocket for PY motif prolines. Extensive contacts are formed between the betaENaC-PY motif and the Pro in WW3*, and the small Ala creates a large pocket to accommodate the peptide. Indeed, mutating the conserved Pro and Ala in WW3* reduces binding affinity 2-3-fold. Additionally, we demonstrate that mutations in PY motif residues that form contacts with the WW domain based on our previously solved structure either abolish or severely reduce binding affinity to the WW domain and that the extent of binding correlates with the level of ENaC suppression. Independently, we show that a peptide encompassing the PY motif of sgk1, previously proposed to bind to Nedd4-2 and alter its ability to regulate ENaC, does not bind (or binds poorly) the WW domains of Nedd4-2. Collectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression by Nedd4 proteins.     

Phosphorylation of a single subunit of the epithelial Na+ channel protein following vasopressin treatment of A6 cells

Arginine vasopressin (antidiuretic hormone, ADH) stimulation of sodium transport in high electrical resistance epithelia is accompanied by adenylate cyclase stimulation and cAMP accumulation. The hypothesis of direct phosphorylation of the purified amiloride-blockable epithelial Na+ channel protein by cAMP-dependent protein kinase A after ADH treatment of cultured cells was investigated in this study. Phosphate-depleted A6 cells (a cell line derived from toad kidney) were exposed to 32PO4(3-) in the absence or presence of basolateral ADH (100 milliunits/ml). After 20 min (the time needed for ADH to increase maximally Na+ transport), the Na+ channels were extracted from the cells and purified. At every stage of purification, only one subunit of the Na+ channel, namely, the 315-kDa subunit, was specifically phosphorylated as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography or scintillation counting. In addition, a polyclonal antibody raised against purified epithelial Na+ channel protein was able to immunoprecipitate the phosphorylated channel protein from a detergent-solubilized fraction of vasopressin-treated A6 cells. This same subunit was also specifically phosphorylated in vitro when the purified Na+ channel protein was incubated with gamma-[32P]ATP and the purified catalytic subunit of the cAMP-dependent protein kinase. Thus, only a single component, the 315-kDa subunit, of the Na+ channel protein complex (which is composed of six subunits) can be phosphorylated both in vivo and in vitro. This subunit is selectively phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a level of 2-3 mol of 32P/mol of protein.     

Capsazepine is a novel activator of the delta subunit of the human epithelial Na+ channel

The amiloride-sensitive epithelial Na+ channel (ENaC) regulates Na+ homeostasis into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. The chemical agents acting on ENaC are, however, largely unknown, except for amiloride and benzamil as ENaC inhibitors. In particular, there are no agonists currently known that are selective for ENaCdelta, which is mainly expressed in the brain. Here we demonstrate that capsazepine, a competitive antagonist for transient receptor potential vanilloid subfamily 1, potentiates the activity of human ENaCdeltabetagamma (hENaCdeltabetagamma) heteromultimer expressed in Xenopus oocytes. The inward currents at a holding potential of -60 mV in hENaCdeltabetagamma-expressing oocytes were markedly enhanced by the application of capsazepine (> or =1 microM), and the capsazepine-induced current was mostly abolished by the addition of 100 microM amiloride. The stimulatory effects of capsazepine on the inward current were concentration-dependent with an EC50 value of 8 microM. Neither the application of other vanilloid compounds (capsaicin, resiniferatoxin, and olvanil) nor a structurally related compound (dopamine) modulated the inward current. Although hENaCdelta homomer was also significantly activated by capsazepine, unexpectedly, capsazepine had no effect on hENaCalpha and caused a slight decrease on the hENaCalphabetagamma current. In conclusion, capsazepine acts on ENaCdelta and acts together with protons. Other vanilloids tested do not have any effect. These findings identify capsazepine as the first known chemical activator of ENaCdelta.     

Phosphatidylinositol 4-phosphate 5-kinase reduces cell surface expression of the epithelial sodium channel (ENaC) in cultured collecting duct cells

Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.     

Conservation of Na+ vs. K+ by the rat cortical collecting duct

Regulation of transport by principal cells of the distal nephron contributes to maintenance of Na(+) and K(+) homeostasis. To assess which of these ions is given a higher priority by these cells, we investigated the upregulation of epithelial Na(+) channels (ENaC) in the rat cortical collecting duct (CCD) during Na depletion with and without simultaneous K depletion. ENaC activity, assessed as whole cell amiloride-sensitive current in split-open tubules, was 260 ± 40 pA/cell in K-repleted but virtually undetectable (3 ± 1 pA/cell) in K-depleted animals. This difference was confirmed biochemically by the reduced amounts of the cleaved forms of both the α-ENaC and γ-ENaC subunits measured in immunoblots. In contrast, in K-depleted rats, simultaneously reducing Na intake did not affect the activity of ROMK channels, assessed as tertiapin-Q-sensitive whole cell currents, in the CCDs. The lack of Na current in K-depleted animals was the result of reduced levels of aldosterone in plasma, rather than a reduced sensitivity to the hormone. However, rats on a low-Na, low-K diet for 1 wk did not excrete more Na than those on a low-Na, control-K diet for the same period of time. Immunoblot analysis indicated increased levels of the thiazide-sensitive NaCl cotransporter and the apical Na-H exchanger NHE3. This suggests that with reduced K intake, Na balance is maintained despite reduced aldosterone and Na(+) channel activity by upregulation of Na(+) transport in upstream segments. Under these conditions, Na(+) transport by the aldosterone-sensitive distal nephron is reduced, despite the low-Na intake to minimize K(+) secretion and urinary K losses.     

Na+ and Ca2+ channel expression in cultured newt retinal pigment epithelial cells: Comparison with neuronal types of ion channels

We cultured retinal pigment epithelial (RPE) cells dissociated from adult newt eye and analyzed their voltage-gated ion channels during culture using whole-cell patch-clamp techniques. The results were compared with those of retinal neurons under identical experimental conditions. After 6–9 days in culture (early stage), > 60% of RPE cells developed voltage-gated Na⁺ and Ca²⁺ channels that were not observed in freshly dissociated RPE cells. The number of cells expressing Na⁺ channels and Na⁺ current density were high after 12–15 days in culture (intermediate stage), while the number of Ca²⁺ channel-expressing cells and Ca²⁺ current density were high after 20–30 days in culture (late stage). The activation voltage of the Na⁺ current in the RPE cells was similar to that in neurons. The voltage dependence of Na⁺ current inactivation was somewhat different between two cell types. The steepness of the inactivation curve tended to be less in cultured RPE cells than in neurons, and the half-inactivation voltage was about −54 mV for the RPE cells and −45 mV for neurons. The Ca²⁺ current expressed in cultured RPE cells was too small to detect without replacement of external Ca²⁺ with Ba²⁺. The Ba²⁺ current, like Ca²⁺ current in neurons, was enhanced by Bay-K 8644 and blocked by nicardipine. These results suggest that the RPE cells, like neurons, expressed L-type Ca²⁺ channels in culture. The possibility that the development of both Na²⁺ and Ca²⁺ channels in cultured RPE cells is a manifestation of the transdifferentiation of RPE cells into neurons is discussed. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 377–390, 1997.