The amiloride-sensitive epithelial sodium channel constitutes the rate-limiting step for sodium reabsorption in epithelial cells that line the distal part of the renal tubule, the distal colon, the duct of several exocrine glands, and the lung. The activity of this channel is upregulated by vasopressin and aldosterone, hormones involved in the maintenance of sodium balance, blood volume and blood pressure. We have identified the primary structure of the alpha-subunit of the rat epithelial sodium channel by expression cloning in Xenopus laevis oocytes. An identical subunit has recently been reported. Here we identify two other subunits (beta and gamma) by functional complementation of the alpha-subunit of the rat epithelial Na+ channel. The ion-selective permeability, the gating properties and the pharmacological profile of the channel formed by coexpressing the three subunits in oocytes are similar to that of the native channel.

The amiloride-sensitive epithelial sodium channel, ENaC, is a heteromultimeric protein made up of three homologous subunits (alpha, beta and gamma) (1,2). In vitro, assembly and expression of functional active sodium channels in the Xenopus oocyte is strictly dependent on alpha-ENaC--the beta and gamma subunits by themselves are unable to induce an amiloride-sensitive sodium current in this heterologous expression system (2). In vivo, ENaC constitutes the limiting step for sodium absorption in epithelial cells that line the distal renal tubule, distal colon and the duct of several exocrine glands. The adult lung expresses alpha, beta and gamma ENaC (3,4), and an amiloride-sensitive electrogenic sodium reabsorption has been documented in upper and lower airways (3-7), but it is not established whether this sodium transport is mediated by ENaC in vivo. We inactivated the mouse alpha-ENaC gene by gene targeting. Amiloride-sensitive electrogenic Na+ transport was abolished in airway epithelia from alpha-ENaC(-/-) mice. Alpha-ENaC(-/-) neonates developed respiratory distress and died within 40 h of birth from failure to clear their lungs of liquid. This study shows that ENaC plays a critical role in the adaptation of the newborn lung to air breathing.

The epithelial amiloride-sensitive sodium channel constitutes the rate limiting step for sodium reabsorbtion by the epithelial lining the distal part of the kidney tubule, the urinary bladder and the distal colon. Reabsorbtion of sodium through this channel, which is regulated by hormones such as aldosterone and vasopressin, is one of the essential mechanisms involved in the regulation of sodium balance, blood volume and blood pressure. Here we isolate a DNA from epithelial cells of rat distal colon and identify it by functional expression of an amiloride-sensitive sodium current in Xenopus oocyte. The deduced polypeptide (698 amino acids) has at least two putative transmembrane segments. Expression of this protein in Xenopus oocytes reconstitutes the functional properties of the highly selective amiloride-sensitive, epithelial sodium channel. The gene encoding this rat sodium channel subunit shares significant sequence similarity with mec-4 and deg-1, members of a family of Caenorhabditis elegans genes involved in sensory touch transduction and, when mutated, neuronal degeneration. We propose that the gene products of these three genes are members of a gene family coding for cation channels.

Sensitivity of blood pressure to dietary salt is a common feature in subjects with hypertension. These features are exemplified by the mendelian disorder, Liddle's syndrome, previously shown to arise from constitutive activation of the renal epithelial sodium channel due to mutation in the beta subunit of this channel. We now demonstrate that this disease can also result from a mutation truncating the carboxy terminus of the gamma subunit of this channel; this truncated subunit also activates channel activity. These findings demonstrate genetic heterogeneity of Liddle's syndrome, indicate independent roles of beta and gamma subunits in the negative regulation of channel activity, and identify a new gene in which mutation causes a salt-sensitive form of human hypertension.

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...