Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits

Members of the DEG/ENaC protein family form ion channels with diverse functions. DEG/ENaC subunits associate as hetero- and homomultimers to generate channels; however the stoichiometry of these complexes is unknown. To determine the subunit stoichiometry of the human epithelial Na+ channel (hENaC), we expressed the three wild-type hENaC subunits (alpha, beta, and gamma) with subunits containing mutations that alter channel inhibition by methanethiosulfonates. The data indicate that hENaC contains three alpha, three beta, and three gamma subunits. Sucrose gradient sedimentation of alphahENaC translated in vitro, as well as alpha-, beta-, and gammahENaC coexpressed in cells, was consistent with complexes containing nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced complexes of similar mass. Our results suggest a novel nine-subunit stoichiometry for the DEG/ENaC family of ion channels.     

A molecular component of the arterial baroreceptor mechanotransducer

Baroreceptor nerve endings detect acute fluctuations in arterial pressure. We tested the hypothesis that members of the DEG/ENaC family of cation channels, which are responsible for touch sensation in Caenorhabditis elegans, may be components of the baroreceptor mechanosensor. We found the gamma subunit of ENaC localized to the site of mechanotransduction in baroreceptor nerve terminals innervating the aortic arch and carotid sinus. A functional role for DEG/ENaC members was suggested by blockade of baroreceptor nerve activity and baroreflex control of blood pressure by an amiloride analog that inhibits DEG/ENaC channels. These data suggest that ENaC subunits may be components of the baroreceptor mechanotransducer and pave the way to a better definition of mechanisms responsible for blood pressure regulation and hypertension.     

Protons activate brain Na+ channel 1 by inducing a conformational change that exposes a residue associated with neurodegeneration

BNC1 is a mammalian neuronal cation channel in the novel DEG/ENaC ion channel family. BNC1 channels are transiently activated by extracellular protons and are constitutively activated by insertion of large residues, such as valine, in place of Gly-430; residue 430 is a site where analogous mutations in some Caenorhabditis elegans family members cause a swelling neurodegeneration. Mutation of Gly-430 to a small amino acid, cysteine, neither generated constitutive currents nor allowed modification of this residue by sulfhydryl-reactive methanethiosulfonate (MTS) compounds. However, when protons activated the channel, Cys-430 became accessible to extracellular MTS reagents, which modified Cys-430 to generate constitutive currents. Fluorescent MTS reagents also labeled Cys-430 in activated channels. These data indicate that protons induce a reversible conformational change that activates BNC1 thereby exposing residue 430 to the extracellular solution. Once Cys-430 is modified with a large chemical group, the channel is prevented from relaxing back to the inactive state. These results link ligand-dependent activation and activation by mutations that cause neurodegeneration.     

Molecular cloning of a DEG/ENaC sodium channel cDNA from human testis

The number of members of the recently defined DEG/ENaC sodium channel superfamily is increasing. Their importance in Na transport, taste perception, acid sensing, and mechanotransduction has been implicated. We have cloned a new member of this superfamily from human testis, which was named hTNaC1 (for human testis sodium channel 1). The hTNaC1 has 532 amino acid residues with two hydrophobic transmembrane domains. It has the highest identity (82%) with a rat H(+)-gated Na channel specific for sensory neurons (DRASIC) and a low identity (29%) with an epithelial isoform (alpha-ENaC) of this superfamily. Northern blot of human tissues revealed its selective expression in testis (7 kb) and absence in other tissues. The identification of a new member of Na channel specifically expressed in testis will expand the role of this channel family to the reproduction physiology.     

An epithelial serine protease activates the amiloride-sensitive sodium channel

Sodium balance, and ultimately blood pressure and extracellular fluid volume, is maintained by precise regulation of the activity of the epithelial sodium channel (ENaC). In a Xenopus kidney epithelial cell line (A6), exposure of the apical membrane to the protease inhibitor aprotinin reduces transepithelial sodium transport. Sodium-channel activity can be restored by subsequent exposure to the nonspecific protease trypsin. Using A6 cells and a functional complementation assay to detect increases in ENaC activity, we have cloned a 329-residue protein belonging to the serine protease family. We show that coexpression of this protein with ENaC in Xenopus oocytes increases the activity of the sodium channel by two- to threefold. This channel-activating protease (CAP1) is expressed in kidney, gut, lung, skin and ovary. Sequence analysis predicts that CAP1 is a secreted and/or glycosylphosphatidylinositol-anchored protein: ENaC activity would thus be regulated by the activity of a protease expressed at the surface of the same cell. This previously undiscovered mechanism for autocrine regulation may apply to other ion channels, in particular to members of the ENaC family that are present in neurons and epithelial cells.     

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.     

Structure and function of the Mec-ENaC family of ion channels

The epithelial sodium channel (ENaC) is the prototype of a new family of ion channels known as the Mec-ENaC superfamily. This new family of proteins are involved in a wide variety of functions that range from maintenance of sodium homeostasis to transduction of mechanical stimuli and nociceptive pain by specialized neurons. They show distinct tissue- and cell type-dependent expression and differential sensitivity to inhibition by the diuretic amiloride and its analogs. Despite the very little amino acid identity shared by these proteins, they all have the same common structure that has become a hallmark of the Mec-ENaC superfamily. The efforts to understand the structure and regulation of these ion channels have been stimulated by the recent discovery of severe disturbances in the maintenance of blood pressure caused by gain- or loss-of-function mutations in the genes that encode the subunits of ENaC in humans. Moreover, cloning of the ion channels that mediate pain elicited by tissue injury and inflammation will facilitate the development of new drugs to treat these common ailments.     

The nematode degenerin UNC-105 forms ion channels that are activated by degeneration- or hypercontraction-causing mutations

Nematode degenerins have been implicated in touch sensitivity and other forms of mechanosensation. Certain mutations in several degenerin genes cause the swelling, vacuolation, and death of neurons, and other mutations in the muscle degenerin gene unc-105 cause hypercontraction. Here, we confirm that unc-105 encodes an ion channel and show that it is constitutively active when mutated. These mutations disrupt different regions of the channel and have different effects on its gating. The UNC-105 channels are permeable to small monovalent cations but show voltage-dependent block by Ca2+ and Mg2+. Amiloride also produces voltage-dependent block, consistent with a single binding site 65% into the electric field. Mammalian cells expressing the mutant channels accumulate membranous whorls and multicompartment vacuoles, hallmarks of degenerin-induced cell death across species.     

dGNaC1, a gonad-specific amiloride-sensitive Na+ channel

Amiloride-sensitive sodium channels have been implicated in reproductive and early developmental processes of several species. These include the fast block of polyspermy in Xenopus oocytes that follows the sperm binding to the egg or blastocoel expansion in mammalian embryo. We have now identified a gene called dGNaC1 that is specifically expressed in the gonads and early embryo in Drosophila melanogaster. The corresponding protein belongs to the superfamily of cationic channels blocked by amiloride that includes Caenorhabditis elegans degenerins, the Helix aspersa FMRF-amide ionotropic receptor (FaNaC), the mammalian epithelial Na+ channel (ENaC), and acid-sensing ionic channels (ASIC, DRASIC, and MDEG). Expression of dGNaC1 in Xenopus oocytes generates a constitutive current that does not discriminate between Na+ and Li+, but is selective for Na+ over K+. This current is blocked by amiloride (IC50 = 24 microM), benzamil (IC50 = 2 microM), and ethylisopropyl amiloride (IC50 = 49 microM). These properties are clearly different from those obtained after expression of the previously cloned members of this family, including ENaC and the human alphaENaC-like subunit, deltaNaC. Interestingly, the pharmacology of dGNaC1 is not very different from that found for the Na+ channel characterized in rabbit preimplantation embryos. We postulate that this channel may participate in gametogenesis and early embryonic development in Drosophila.     

Pioneer axon guidance by UNC-129, a C. elegans TGF-beta

The unc-129 gene, like the unc-6 netrin gene, is required to guide pioneer motoraxons along the dorsoventral axis of Caenorhabditis elegans. unc-129 encodes a member of the transforming growth factor-beta (TGF-beta) superfamily of secreted signaling molecules and is expressed in dorsal, but not ventral, rows of body wall muscles. Ectopic expression of UNC-129 from ventral body wall muscle disrupts growth cone and cell migrations that normally occur along the dorsoventral axis. Thus, UNC-129 mediates expression of dorsoventral polarity information required for axon guidance and guided cell migrations in C. elegans.     

Pore stoichiometry of a voltage-gated chloride channel

Ion channels allow ions to pass through cell membranes by forming aqueous permeation pathways (pores). In contrast to most known ion channels, which have single pores, a chloride channel belonging to the CIC family (Torpedo CIC-0) has functional features that suggest that it has a unique 'double-barrelled' architecture in which each of two subunits forms an independent pore. This model is based on single-channel recordings of CIC-0 that has two equally spaced and independently gated conductance states. Other CIC isoforms do not behave in this way, raising doubts about the applicability of the model to all CIC channels. Here we determine the pore stoichiometry of another CIC isoform, human CIC-1, by chemically modifying cysteines that have been substituted for other amino acids located within the CIC ion-selectivity filter. The CIC-1 channel can be rendered completely susceptible to block by methanethiosulphonate reagents when only one of the two subunits contains substituted cysteines. Thiol side chains placed at corresponding positions in both subunits can form intersubunit disulphide bridges and coordinate Cd2+, indicating that the pore-forming regions from each subunit line the same conduction pathway. We conclude that human CIC-1 has a single functional pore.     

Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain

The generation of pacemaker activity in heart and brain is mediated by hyperpolarization-activated cation channels that are directly regulated by cyclic nucleotides. We previously cloned a novel member of the voltage-gated K channel family from mouse brain (mBCNG-1) that contained a carboxy-terminal cyclic nucleotide-binding domain (Santoro et al., 1997) and hence proposed it to be a candidate gene for pacemaker channels. Heterologous expression of mBCNG-1 demonstrates that it does indeed code for a channel with properties indistinguishable from pacemaker channels in brain and similar to those in heart. Three additional mouse genes and two human genes closely related to mBCNG-1 display unique patterns of mRNA expression in different tissues, including brain and heart, demonstrating that these channels constitute a widely expressed gene family.     

A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP

Extracellular ATP exerts its effects through P2 purinoceptors: these are ligand-gated ion channels (P2x) or G-protein-coupled receptors (P2Y, P2U). ATP at P2x receptors mediates synaptic transmission between neurons and from neurons to smooth muscle, being responsible, for example, for sympathetic vasoconstriction in small arteries and arterioles. We have now cloned a complementary DNA encoding the P2x receptor from rat vas deferens and expressed it in Xenopus oocytes and mammalian cells. ATP activates a cation-selective ion channel with relatively high calcium permeability. Structural predictions suggest that the protein (399 amino acids long) is mostly extracellular and contains only two transmembrane domains plus a pore-forming motif which resembles that of potassium channels. The P2x receptor thus defines a new family of ligand-gated ion channels.     

Excitotoxic death of a subset of embryonic rat motor neurons in vitro

We have used cultures of purified embryonic rat spinal cord motor neurons to study the neurotoxic effects of prolonged ionotropic glutamate receptor activation. NMDA and non-NMDA glutamate receptor agonists kill a maximum of 40% of the motor neurons in a concentration- and time-dependent manner, which can be blocked by receptor subtype-specific antagonists. Subunit-specific antibodies stain all of the motor neurons with approximately the same intensity and for the same repertoire of subunits, suggesting that the survival of the nonvulnerable population is unlikely to be due to the lack of glutamate receptor expression. Extracellular Ca2+ is required for excitotoxicity, and the route of entry initiated by activation of non-NMDA, but not NMDA, receptors is L-type Ca2+ channels. Ca2+ imaging of motor neurons after application of specific glutamate receptor agonists reveals a sustained rise in intracellular Ca2+ that is present to a similar degree in most motor neurons, and can be blocked by appropriate receptor/channel antagonists. Although the lethal effects of glutamate receptor agonists are seen in only a subset of cultured motor neurons, the basis of this selectivity is unlikely to be simply the glutamate receptor phenotype or the level/pattern of rise in agonist-evoked intracellular Ca2+.     

Phenotype variation and newcomers in ion channel disorders

Ion channels are part of a large family of macromolecules whose functions include the control and maintenance of electrical potential across cell membranes, secretion and signal transduction. Close inspection of the physiological processes involved in channel function and the secondary structure of various ion channels has served as a basis for subdividing ion channels into a number of superfamilies. The voltage-gated ion channels are one of these superfamilies. Recent work has shown that mutations in various ion channel genes are responsible for a number of neuromuscular and neurological disorders. Correlation of the various mutations with the clinical phenotype is providing us with insight into the pathophysiology of these channel proteins. Interestingly, different mutations within the same gene may cause quite distinct clinical disorders, while mutations in different channel genes may result in very similar phenotypes (genetic heterogeneity). Examples of phenotypic variation and genetic heterogeneity are presented in the context of the periodic paralytic disorders of skeletal muscle, episodic ataxia, migraine, long QT syndrome and paroxysmal dyskinesia. Some of these disorders are known to be caused by mutations in ion channel genes, while in the episodic movement disorders, ion channel genes are considered excellent candidate genes.     

Subunit stoichiometry of the epithelial sodium channel

The epithelial Na+ Channel (ENaC) mediates Na+ reabsorption in a variety of epithelial tissues. ENaC is composed of three homologous subunits, termed alpha, beta, and gamma. All three subunits participate in channel formation as the absence of any one subunit results in a significant reduction or complete abrogation of Na+ current expression in Xenopus oocytes. To determine the subunit stoichiometry, a biophysical assay was employed utilizing mutant subunits that display significant differences in sensitivity to channel blockers from the wild type channel. Our results indicate that ENaC is a tetrameric channel with an alpha2 beta gamma stoichiometry, similar to that reported for other cation selective channels, such as Kv, Kir, as well as voltage-gated Na+ and Ca2+ channels that have 4-fold internal symmetry.