Liddle's Mutations Research Articles

Disease causing mutations affect ENaC gating

Gain and loss‐of‐function mutations in the epithelial Na channel (ENaC) affect renal Na+ excretion. Here, we identify five mutations in ENaC that lead to changes in single channel properties, and should be informative about structure‐function relations within the channel protein. Channels containing the PHA‐I mutation, G37S in β‐ENaC, are voltage‐dependent where Po is low at physiological potentials. This critical Gly is in a conserved HG motif in the cytosolic NH2‐terminus key to gating. Another PHA‐I mutation, the KYS106‐108‐>N substitution in γ‐ENaC, also markedly decreases current by decreasing Po in a voltage‐dependent manner. The PHA‐I mutation, S562P in γ‐ENaC, results in complete loss of function. S562 occupies a critical position in the selectivity filter of the channel pore possibly being involved in coordination of the conductive ion during permeation. The N530S Liddle's mutation in the γ‐subunit of ENaC replaces a conserved (in ENaC) Arg within the extracellular vestibule of the pore one position downstream of the Deg site. Channels containing γ‐ENaC harboring the N530S substitution have increased Po. The Liddle's mutation W493R in α‐ENaC increases channel activity and Po. W493 also is located in extracellular mouth of the pore and is believed to interact in an electrostatic manner with the key pore residue R492. Notabely, none of these mutations affected single channel conductance.

Stimulation of ENaC by Thiasolidinediones

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists used to treat type II diabetes. Recent studies have shown that TZDs can cause fluid retention and oedema by increasing sodium reabsorption in the renal collecting duct. Those side effects may be caused by the up-regulation of the epithelial Na+ channel (ENaC) and Na+/K+-ATPase. However, the mechanisms involved are not clearly understood. Our goal is to study the effects and the mechanisms involved in rosiglitazone action on the expression and function of ENaC. To do so, we performed two electrode voltage clamp studies (TEVC) in Xenopus laevis oocytes expressing PPARγ receptor, wild type (wt) and mutant ENaC channels. We have shown that a 48h treatment with 10 μM RGZ produced a 2-fold increase of ENaC activity and this activation is blocked by GW9662, a PPARγ antagonist. We have also generated a mutation in a potential SGK1 (serum- and glucocorticoid-regulated kinase) binding site in the ENaC subunit and expressed the mutant channel together with the PPARγ receptor in Xenopus oocytes. RGZ-induced activation was similar in both wt and mutant channels, suggesting that direct phosphorylation of ENaC by SGK1 is not involved in this regulation. SGK1 is also known to inhibit ENaC internalization through Nedd4-2 phosphorylation and subsequent inactivation. ENaC lacking Nedd4-2 binding motifs (Liddle's mutation) are not stimulated by RGZ. Those results suggest RGZ treatment have increased ENaC expression and activity through Nedd4-2 inhibition mediated by SGK1. In accordance with these results, RGZ increase the activity of ENaC by enhancing its cell surface expression, most probably indirectly, through the increase of SGK1 expression. Western blot analysis and confocal microscopy experiments have confirmed that the RGZ-induced ENaC activity and expression via the SGK1/Nedd4-2 pathway in our model.

Pendrin Regulation in Mouse Kidney Primarily Is Chloride-Dependent

Recent studies indicate that pendrin, an apical Cl-/HCO3- exchanger, mediates chloride reabsorption in the connecting tubule and the cortical collecting duct and therefore is involved in extracellular fluid volume regulation. The purpose of this study was to test whether pendrin is regulated in vivo primarily by factors that are associated with changes in renal chloride transport, by aldosterone, or by the combination of both determinants. For achievement of this goal, pendrin protein abundance was studied by semiquantitative immunoblotting in different mouse models with altered aldosterone secretion or tubular chloride transport, including NaCl loading, hydrochlorothiazide administration, NaCl co-transporter knockout mice, and mice with Liddle's mutation. The parallel regulation of the aldosterone-regulated epithelial sodium channel (ENaC) was examined as a control for biologic effects of aldosterone. Major changes in pendrin protein expression were found in experimental models that are associated with altered renal chloride transport, whereas no significant changes were detected in pendrin protein abundance in models with altered aldosterone secretion. Moreover, in response to hydrochlorothiazide administration, pendrin was downregulated despite a marked secondary hyperaldosteronism. In contrast, alpha-ENaC was markedly upregulated, and the molecular weight of a large fraction of gamma-ENaC subunits was shifted from 85 to 70 kD, consistent with previous results from rat models with elevated plasma aldosterone levels. These results suggest that factors that are associated with changes in distal chloride delivery govern pendrin expression in the connecting tubule and cortical collecting duct.

Overexpression of the Epithelial Na+ Channel γ Subunit in Collecting Duct Cells: INTERACTIONS OF LIDDLE'S MUTATIONS AND STEROIDS ON EXPRESSION AND FUNCTION

The epithelial Na(+) channel (ENaC) has three subunits; the expression of each can be regulated. Liddle's syndrome is caused by an activating mutation in the C terminus of either the beta or gamma subunit. We used a doxycycline-regulated adenovirus system to express varying levels of human gammaENaC in renal collecting duct (M1 cell) monolayers. Increasing levels of wild type human gamma ENaC (gammahENaC) produced a 2.5-fold enhancement of Na(+) transport. Expression of a truncated C terminus produced less protein than wild type or a gammaY627A missense mutation. However, either of these mutations produced a approximately 4-fold increase in Na(+) transport despite the different levels of protein expression. Unexpectedly, overexpression of a marginally detectable amount of gammahENaC was sufficient to produce a full increase in Na(+) transport; a further increase in protein expression produced no further increase in Na(+) transport. Steroid treatment increased Na(+) transport to a similar absolute magnitude in control monolayers and in monolayers expressing all types of gammahENaC. Withdrawal of steroids after 24 h produced a decline in Na(+) transport over 8 h in monolayers expressing wild type but not the Liddle's mutation. Using treatment with brefeldin A to estimate the disappearance rate constants, we found progressively slower disappearance rates in monolayers overexpressing gammahENaC and the Liddle's mutant. Calculated insertion rates were slower for the Liddle's mutant than for wild type despite increasing rates of Na(+) transport. These results raise questions regarding previously held assumptions about the behavior of ENaC.

The Activity of the Epithelial Sodium Channel Is Regulated by Clathrin-mediated Endocytosis

Activity of the epithelial sodium channel (ENaC) is a key determinant of sodium homeostasis and blood pressure. Liddle's syndrome, an inherited form of hypertension, is caused by mutations that delete or alter PY domains in the carboxyl termini of beta or gamma ENaC subunits, leading to increased channel activity. In this study we investigated the mechanism of this effect by analysis of wild-type and mutant ENaC activity in Xenopus oocytes. By inhibiting insertion of new channels into the plasma membrane with brefeldin A, we demonstrate that the half-life of the activity of channels containing Liddle's mutations is markedly prolonged compared with wild-type channels (t1/2 of 30 h in mutant versus 3.6 in wild-type, p < 0.001). We investigated the involvement of clathrin-coated pit-mediated endocytosis by co-expressing a dominant-negative dynamin mutant with wild-type ENaC in oocytes. Expression of this specific inhibitor of endocytosis leads to a large increase in the activity of wild-type channels, demonstrating that normal turnover of this channel is through the clathrin-coated pit pathway. In contrast, co-expression of Liddle's mutations and dynamin mutants leads to no further increase in channel activity, consistent with one of the effects of Liddle's mutations being the loss of endocytosis of these channels. These findings demonstrate the normal mechanism of turnover of ENaC from the cell surface and demonstrate a mechanism that can account for the increased number of channels in the plasma membrane seen in Liddle's syndrome.

Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na + channel

Liddle's syndrome is an inherited form of hypertension caused by mutations that truncate the C-terminus of human epithelial Na + channel (hENaC) subunits. Expression of truncated β and γ hENaC subunits increased Na + current. However, truncation did not alter single-channel conductance or open state probability, suggesting there were more channels in the plasma membrane. Moreover, truncation of the C-terminus of the β subunit increased apical cell-surface expression of hENaC in a renal epithelium. We identified a conserved motif in the C-terminus of all three subunits that, when mutated, reproduced the effect of Liddle's truncations. Further, both truncation of the C-terminus and mutation of the conserved C-terminal motif increased surface expression of chimeric proteins containing the C-terminus of β hENaC. Thus, by deleting a conserved motif, Liddle's mutations increase the number of Na + channels in the apical membrane, which increases renal Na + absorption and creates a predisposition to hypertension.