Regulation Of Epithelial Sodium Channel Research Articles

Pathways for Camp Regulation of Epithelial Sodium Channels in Epithelial Cells: Possible Crosstalk between Pka and Sgk1 Pathways

Pathways for Camp Regulation of Epithelial Sodium Channels in Epithelial Cells: Possible Crosstalk between Pka and Sgk1 Pathways

All Three WW Domains of Murine Nedd4 Are Involved in the Regulation of Epithelial Sodium Channels by Intracellular Na+

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PY motif of either the beta or gamma subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind alpha-, beta-, and gamma-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+ channel and of WW domain 1 to an unknown associated protein.

Accessory factors and the regulation of epithelial sodium channel activity.

The contribution in this issue of the JCI by Abriel et al. (1) brings into sharp focus our expanding knowledge of the regulation of epithelial sodium channel (ENaC) activity by specific accessory proteins. It has long been recognized that interactions with cytoskeletal proteins, such as actin, can regulate ENaC in a number of model systems (2). The interactions with defined cytosolic regions of the ENaC subunits were demonstrated by earlier studies of the ubiquitin ligase Nedd4 by Staub et al. (3). The importance of this interaction was emphasized by the finding that the site of interaction was with specific proline-rich regions of the subunits and that these very sites were found to be mutated, or even missing, in patients with Liddle's syndrome (4). This autosomal dominant syndrome is a rare cause of human hypertension that has clearly been shown to result from the failure to properly regulate ENaC expression and activity, with volume-expanded low-renin hypertension as the direct consequence. It appears that Nedd4 negatively modulates ENaC activity; binding of its WW domains to the proline domains of ENaC is followed by ubiquitination of the channel subunits, with subsequent endocytosis and lysosomal degradation. Indeed, ENaC appears to turn over quite rapidly with critical NH2-terminal lysine residues identified as the sites of ubiquitination of the α and γ subunits (5). In the current studies, the Xenopus oocyte expression system was used to demonstrate that overexpression of Nedd4 with ENaC inhibited channel activity. This effect was critically dependent on the proline domains in the ENaC subunits and on intact ubiquitination activity of the Nedd4 protein (1). Of note, a catalytically inactive Nedd4 construct appeared to interact competitively with the wild-type Nedd4 protein and actually protected the expressed ENaC from ubiquitination. A similar dependence of the regulatory effect of Nedd4 on its COOH-terminal ubiquitin ligase domain was recently demonstrated by Goulet et al. (6). These changes in ENaC activity can be rationalized in terms of changes in the surface expression of the channel complex, consistent with the role of the Nedd4 protein in endocytosis and degradation of the assembled ENaC complex at the surface membrane. While these effects are clearly explained by changes in the surface expression the ENaC complexes, there also appear to be direct kinetic effects of the various mutations described in Liddle's syndrome on the apparent open probability of the expressed ENaC (7). Certainly, endocytosis plays an important role in determining channel density or dwell time in the surface membrane, but other factors may also affect the rate of endocytosis (8), and even exocytosis, of the assembled channel complex to the surface membrane (9, 10). Other factors, including K-Ras2A, a small G protein that may in fact be one of the long-sought aldosterone-induced proteins (11), and an ENaC-associated serine protease termed channel activating protein-1(12) can activate ENaC activity independently of changes in its surface expression. In fact, K-Ras2A markedly increases in ENaC activity despite a decrease in surface expression (11), while we find that syntaxin 1A increases ENaC surface expression but decreases functional ENaC activity (9). This ensemble of studies has revealed important themes and details of the short-term regulation of ENaC activity. Although these studies have relied heavily on the Xenopus oocyte expression system, it can be expected that these results will provide novel approaches to the understanding, and even therapy, of human disorders of ENaC regulation. As the panoply of accessory proteins and factors unfolds, each provides a new candidate for the exploration of the functional regulation of ENaC activity, and even potential genetic linkage approaches to defined subsets of human low-renin hypertension.

Role of actin in regulation of epithelial sodium channels by CFTR.

Cystic fibrosis (CF) airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that actin plays a role in the regulation of a cloned epithelial Na+ channel (ENaC) by the cystic fibrosis transmembrane conductance regulator (CFTR). We found that immunopurified bovine tracheal CFTR coreconstituted into a planar lipid bilayer with alpha,beta,gamma-rat ENaC (rENaC) decreased single-channel open probability (Po) of rENaC in the presence of actin by over 60%, a significantly greater effect than was observed in the absence of actin (approximately 20%). In the presence of actin, protein kinase A plus ATP activated both CFTR and rENaC, but CFTR was activated in a sustained manner, whereas the activation of rENaC was transitory. ATP alone could also activate ENaC transiently in the presence ofactin but had no effect on CFTR. Stabilizing short actin filaments at a fixed length with gelsolin (at a ratio to actin of 2:1) produced a sustained activation of alpha,beta,gamma-rENaC in both the presence or absence of CFTR. Gelsolin alone (i.e., in the absence of actin) had no effect on the conductance or Po of either CFTR or rENaC. We have also found that short actin filaments produced their modulatory action on alpha-rENaC independent of the presence of the beta- or gamma-rENaC subunits. In contrast, CFTR did not affect any properties of the channel formed by alpha-rENaC alone, i.e., in the absence of beta- or gamma-rENaC. These results indicate that CFTR can directly downregulate single Na+ channel activity, which may account for the observed differences between Na+ transport in normal and CF-affected airway epithelia. Moreover, the presence of actin confers an enhanced modulatory ability of CFTR on Na+ channels.

Regulation of Epithelial Sodium Channels by Short Actin Filaments

Cytoskeletal elements play an important role in the regulation of ion transport in epithelia. We have studied the effects of actin filaments of different length on the alpha, beta, gamma-rENaC (rat epithelial Na+ channel) in planar lipid bilayers. We found the following. 1) Short actin filaments caused a 2-fold decrease in unitary conductance and a 2-fold increase in open probability (Po) of alpha,beta,gamma-rENaC. 2) alpha,beta,gamma-rENaC could be transiently activated by protein kinase A (PKA) plus ATP in the presence, but not in the absence, of actin. 3) ATP in the presence of actin was also able to induce a transitory activation of alpha, beta,gamma-rENaC, although with a shortened time course and with a lower magnitude of change in Po. 4) DNase I, an agent known to prohibit elongation of actin filaments, prevented activation of alpha,beta,gamma-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of alpha,beta,gamma-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of alpha,beta,gamma-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of alpha,beta,gamma-rENaC at actin/gelsolin ratios of <32:1, with a maximal effect at an actin/gelsolin ratio of 2:1. These results suggest that short actin filaments activate alpha, beta,gamma-rENaC. PKA-mediated phosphorylation augments activation of this channel by decreasing the rate of elongation of actin filaments. These results are consistent with the hypothesis that cloned alpha,beta,gamma-rENaCs form a core conduction unit of epithelial Na+ channels and that interaction of these channels with other associated proteins, such as short actin filaments, confers regulation to channel activity.

Regulation of Epithelial Sodium Channels by the Cystic Fibrosis Transmembrane Conductance Regulator

Cystic fibrosis airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) directly affects epithelial Na+ channel activity by co-incorporating into planar lipid bilayers immunopurified bovine tracheal CFTR and either heterologously expressed rat epithelial Na+ channel ( alpha,b eta,gamma-rENaC) or an immunopurified bovine renal Na+ channel protein complex. The single channel open probability (Po) of rENaC was decreased by 24% in the presence of CFTR. Protein kinase A (PKA) plus ATP activated CFTR, but did not have any effect on rENaC. CFTR also decreased the extent of elevation of the renal Na+ channel Po following PKA-mediated phosphorylation. Moreover, the presence of CFTR prohibited the inward rectification of the gating of this renal Na+ channel normally induced by PKA-mediated phosphorylation, thus down-regulating inward Na+ current. This interaction between CFTR and Na+ channels occurs independently of whether or not wild-type CFTR is conducting anions. However, the nonconductive CFTR mutant, G551D CFTR, cannot substitute for the wild-type molecule. Our results indicate that CFTR can directly down-regulate single Na+ channel activity, thus accounting, at least in part, for the observed differences in Na+ transport between normal and cystic fibrosis-affected airway epithelia.