The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na ؉ movement in Na ؉ -transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surfaceexpressed ENaC. We have developed a quantitative assay based on the binding of 125 I-labeled M 2 anti-FLAG monoclonal antibody (M 2 Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the ␣, ␤, and ␥ ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (K d ‫؍‬ 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (I Na ) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of ␣, ␤, and ␥ ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (␤ R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.The amiloride-sensitive epithelial sodium channel (ENaC) is a heteromultimeric protein composed of three homologous subunits, ␣, ␤, and ␥ (1, 2, 4, 5), exhibiting Ϸ30% identity at the amino acid level. Predicted protein topology reveals a large (Ϸ500 amino acids) extracellular hydrophilic loop with several putative N-linked glycosylation sites flanked by two hydrophobic domains (M1 and M2) that span the membrane.In aldosterone target epithelia, ENaC represents the ratelimiting step for Na ϩ reabsorption (6, 7). The control of Na ϩ movements in these epithelia is critical for the maintenance of extracellular fluid and electrolyte balance, and the central role of ENaC in this regulation is exemplified by the recent discoveries of several heritable human mutations in the genes encoding the ENaC subunits that lead to abnormal regulation of blood pressure and electrolyte balance (8, 9). Therefore, identification of the molecular and cellular mechanisms involved in the regulation of ENaC channel activity at the cell surface is critical for our understanding of the pathogenesis of salt-sensitive hypertension. ENaC is characterized by its high ionic selectivity for sodium and lithium, by its low single-channel conductance (5 pS in the presence of Na and 8 pS in the presence of Li), by its long open and closed times, and by its high affinity for amiloride. Our knowled...

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (K i) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride K i by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn2+ ions, in particular the αS583C mutant showing a K i for Zn2+of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride K i, the later mutation generating a Ca2+blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ions at an external Na+binding site preventing ion permeation through the channel pore.

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (α, β and γ) assemble to form a highly Na ϩ -selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC α, β and γ subunits shows that they assemble according to a fixed stoichiometry, with α ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each α, β and γ subunit are consistent with a four subunit stoichiometry composed of two α, one β and one γ. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two α subunits separated by β and γ subunits.

Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. This functional periodicity is believed to result, at least in part, from circadian changes in secretion/reabsorption capacities of the distal nephron and collecting ducts. Here, we studied the molecular mechanisms underlying circadian rhythms in the distal nephron segments, i.e., distal convoluted tubule (DCT) and connecting tubule (CNT) and the cortical collecting duct (CCD). Temporal expression analysis performed on microdissected mouse DCT/CNT or CCD revealed a marked circadian rhythmicity in the expression of a large number of genes crucially involved in various homeostatic functions of the kidney. This analysis also revealed that both DCT/CNT and CCD possess an intrinsic circadian timing system characterized by robust oscillations in the expression of circadian core clock genes (clock, bma11, npas2, per, cry, nr1d1) and clock-controlled Par bZip transcriptional factors dbp, hlf, and tef. The clock knockout mice or mice devoid of dbp/hlf/tef (triple knockout) exhibit significant changes in renal expression of several key regulators of water or sodium balance (vasopressin V2 receptor, aquaporin-2, aquaporin-4, ␣ENaC). Functionally, the loss of clock leads to a complex phenotype characterized by partial diabetes insipidus, dysregulation of sodium excretion rhythms, and a significant decrease in blood pressure. Collectively, this study uncovers a major role of molecular clock in renal function.circadian rhythm ͉ homeostasis ͉ renal function R ecent evidence suggests that many if not all specific physiological functions are under the control of the circadian timing system. The mammalian circadian timing system is a hierarchically organized network of molecular oscillators driven by a central pacemaker located in the suprachiasmatic nucleus (SCN) of hypothalamus. This central pacemaker functions in a self-sustained fashion, but is reset each day by exposure to environmental synchronizers, mainly the light/dark cycle. The SCN masterclock drives the rest-activity cycle, which in turn imposes the feeding pattern [reviewed in (1, 2)]. The feeding time seems to be the dominant cue for circadian rhythms in the peripheral tissues (3, 4). Central and peripheral oscillators share a similar molecular core clock based on a set of self-autonomous transcriptional/ translational feedback loops. The key molecular components of these loops are the PAS domain transcriptional factors CLOCK, BMAL1, and NPAS2 and the feedback repressors PER1, PER2, CRY1, and CRY2. The orphan nuclear receptors NR1D1 and, probably, NR1D2 form an accessory feedback loop. The core oscillators confer circadian rhythmicity on a set of output genes underlying the tissue-specific functional rhythms. Current estimates indicate that up to 10% of the cellular transcriptome may follow a circadian expression pattern (5-7). Several recent studies have also demonstrated that the transcription of only a minority of these circadian genes is driven by systemic humoral or neurona...

Elevated plasma urate levels are associated with metabolic, cardiovascular, and renal diseases. Urate may also form crystals, which can be deposited in joints causing gout and in kidney tubules inducing nephrolithiasis. In mice, plasma urate levels are controlled by hepatic breakdown, as well as, by incompletely understood renal processes of reabsorption and secretion. Here, we investigated the role of the recently identified urate transporter, Glut9, in the physiological control of urate homeostasis using mice with systemic or liver-specific inactivation of the Glut9 gene. We show that Glut9 is expressed in the basolateral membrane of hepatocytes and in both apical and basolateral membranes of the distal nephron. Mice with systemic knockout of Glut9 display moderate hyperuricemia, massive hyperuricosuria, and an early-onset nephropathy, characterized by obstructive lithiasis, tubulointerstitial inflammation, and progressive inflammatory fibrosis of the cortex, as well as, mild renal insufficiency. In contrast, liver-specific inactivation of the Glut9 gene in adult mice leads to severe hyperuricemia and hyperuricosuria, in the absence of urate nephropathy or any structural abnormality of the kidney. Together, our data show that Glut9 plays a major role in urate homeostasis by its dual role in urate handling in the kidney and uptake in the liver.gout ͉ knockout ͉ nephrolithiasis ͉ uric acid