Chloride channels have several functions, including the regulation of cell volume, stabilizing membrane potential, signal transduction and transepithelial transport. The plasma membrane Cl- channels already cloned belong to different structural classes: ligand-gated channels, voltage-gated channels, and possibly transporters of the ATP-binding-cassette type (if the cystic fibrosis transmembrane regulator is a Cl- channel). The importance of chloride channels is illustrated by the phenotypes that can result from their malfunction: cystic fibrosis, in which transepithelial transport is impaired, and myotonia, in which ClC-1, the principal skeletal muscle Cl- channel, is defective. Here we report the properties of ClC-2, a new member of the voltage-gated Cl- channel family. Its sequence is approximately 50% identical to either the Torpedo electroplax Cl- channel, ClC-0 (ref. 8), or the rat muscle Cl- channel, ClC-1 (ref. 9). Isolated initially from rat heart and brain, it is also expressed in pancreas, lung and liver, for example, and in pure cell lines of fibroblastic, neuronal, and epithelial origin, including tissues and cells affected by cystic fibrosis. Expression in Xenopus oocytes induces Cl- currents that activate slowly upon hyperpolarization and display a linear instantaneous current-voltage relationship. The conductivity sequence is Cl- greater than or equal to Br- greater than I-. The presence of ClC-2 in such different cell types contrasts with the highly specialized expression of ClC-1 (ref. 9) and also with the cloned cation channels, and suggests that its function is important for most cells.

Autosomal recessive pseudohypoaldosteronism type I is a rare life-threatening disease characterized by severe neonatal salt wasting, hyperkalaemia, metabolic acidosis, and unresponsiveness to mineralocorticoid hormones. Investigation of affected offspring of consanguineous union reveals mutations in either the alpha or beta subunits of the amiloride-sensitive epithelial sodium channel in five kindreds. These mutations are homozygous in affected subjects, co-segregate with the disease, and introduce frameshift, premature termination or missense mutations that result in loss of channel activity. These findings demonstrate the molecular basis and explain the pathophysiology of this disease.

Acid-sensing ion channels (ASICs) constitute a branch of the super-gene family of amiloride-sensitive sodium channels. So far five different ASICs have been cloned from mammalian tissues. They are activated by a drop of extracellular pH but differ with respect to effective agonist concentration, desensitization and mRNA expression pattern. Here we report cloning of ASIC4, a new protein showing about 45% identity to other ASICs. ASIC4 is 97% identical between rat and human and shows strongest expression in pituitary gland. Moreover, we detected expression throughout the brain, in spinal cord, and inner ear. ASIC4 cannot be activated by a drop of extracellular pH in Xenopus oocytes, suggesting association with other subunits or activation by a ligand different from protons. Our results suggest a role for ASICs also in endocrine glands.

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel distal nephron, the distal colon, the salivary and sweat Stefan Grü nder, Dmitri Firsov, glands and the lung. In these polarized epithelia, the Sue S.Chang 1 , Nicole Fowler Jaeger, ENaC-mediated (Canessa et al., 1993(Canessa et al., , 1994b. Each subunit has two transmembrane domains with short cytoplasmic NPseudohypoaldosteronism type 1 (PHA-1) is an inherand C-termini and a large extracellular loop (Canessa ited disease characterized by severe neonatal salt et al., 1994a;Renard et al., 1994;Snyder et al., 1994). wasting and caused by mutations in subunits of the The biophysical properties and the pharmacological profile amiloride-sensitive epithelial sodium channel (ENaC).of ENaC expressed in Xenopus oocytes are similar to A missense mutation (G37S) of the human ENaC β those for the native channel in the distal nephron (Palmer subunit that causes loss of ENaC function and PHA- 1 and Frindt, 1986;Canessa et al., 1994b). replaces a glycine that is conserved in the N-terminusThe importance of ENaC for the regulation of sodium of all members of the ENaC gene family. We now balance, blood volume and blood pressure has been report an investigation of the mechanism of channel demonstrated by the finding of mutations in either the inactivation by this mutation. Homologous mutations, β or the γ subunits that cause increased ENaC activity introduced into α, β or γ subunits, all significantly and Liddle's disease, a rare, inherited form of salt-sensitive reduce macroscopic sodium channel currents recorded hypertension (Shimkets et al., 1994;Hansson et al., 1995). in Xenopus laevis oocytes. Quantitative determination Recently, it has been shown that pseudohypoaldosteronism of the number of channel molecules present at the cell type 1 (PHA-1), an inherited disease characterized by surface showed no significant differences in surface severe neonatal salt-wasting, hyperkalaemia, metabolic expression of mutant compared with wild-type chanacidosis and unresponsiveness to mineralocorticoid hornels. Single channel conductances and ion selectivities mones, is also caused by mutations in ENaC subunits of the mutant channels were identical to that of wild- (Chang et al., 1996), further confirming the important role type. These results suggest that the decrease in macroof ENaC for salt and water homeostasis. These mutations scopic Na currents is due to a decrease in channel lead either to a frameshift or a premature stop codon in open probability (P o ), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits the α subunit or to an amino acid exchange in a conserved change ENaC channel gating, which would explain the region at the N-terminus of the β subunit (Chang et al., disease pathophysiology. Single channel recordings of 1996). The functional consequences of this mutation in channels containing the mutant α subunit (αG95S) the β subunit (G37S) were inve...

IntroductionType I pseudohypoaldosteronism (PHA-I) is an inherited disorder that manifests soon after birth and is characterized by failure to thrive, salt wasting, metabolic acidosis, life-threatening hyperkalemia, and dehydration. Despite clinical evidence for hypoaldosteronism, plasma aldosterone and plasma renin activity are elevated, suggesting a defect in aldosterone target tissues. Two kinds of inheritance have been described for PHA-I, with distinct clinical and pathophysiological features (1): the autosomal dominant renal form (OMIM 177735), which was recently linked to mutations in the mineralocorticoid receptor gene (2); and the recessive systemic form (OMIM 264350), which presents with severe Na + transport defects in all aldosterone target tissues. It is caused by diminution-of-function mutations in genes encoding the subunits of the amiloride-sensitive epithelial Na + channel (ENaC) (3).ENaC constitutes the rate-limiting step of Na + reabsorption through aldosterone-responsive epithelia found in kidneys, colon, salivary and sweat glands, and glucocorticoid-responsive lung epithelia. ENaC is a heteromultimeric protein made of 3 subunits (α, β, and γ), sharing 35% homology at the protein level and the same membrane topology (4). A heterotetrameric structure (αβαγ) has recently been proposed for ENaC (5,6). It is therefore likely that all cation channels belonging to this gene family are tetrameric, even though a nonameric architecture has been proposed by others (7). Mutagenic studies identified amino acid residues involved in ion permeation and critical for channel block by amiloride in a short segment preceding the second transmembrane domain (the pre-M2 segment) of each of the 3 subunits (8). This demonstrated that all 3 subunits contribute to the formation of the conductive pore. The α subunit plays a specific role in channel assembly and/or targeting to the plasma membrane. This conclusion is based on earlier observations in the Xenopus oocyte system showing that only complexes containing the α subunit (α alone or αβ or αγ complexes) are able to form functional channels. When only βγ subunits were present, no detectable amiloride-sensitive current could be measured (4). Taken together, these data suggest that the α subunit contains some specific information needed to get functional channels to the cell surface -information that is lacking in either β or γ subunits.Homozygous mutations have been identified in all 3 ENaC subunits, causing the recessive form of PHA-I (3). Three mutations in the α subunit have been described: a frameshift (α I68fr ), a missense mutation of a conserved cysteine (α C133Y ), and a premature stop codon (α R508stop ). Truncation of the α subunit is predicted to abolish ENaC activity in all tissues because it The autosomal recessive form of type I pseudohypoaldosteronism (PHA-I) is an inherited salt-losing syndrome resulting from diminution-of-function mutations in the 3 subunits of the epithelial Na + channel (ENaC). A PHA-I stop mutation (α R508stop ) of the ENaC α subu...