The second hydrophobic domain contributes to the kinetic properties of epithelial sodium channels

The epithelial sodium channel (ENaC) is the prototype of a new class of ion channels known as the ENaC/Deg family. The hallmarks of ENaC are a high selectivity for Na(+), block by amiloride, small conductance, and slow kinetics that are voltage-independent. We have investigated the contribution of the second hydrophobic domain of each of the homologous subunits alpha, beta, and gamma to the kinetic properties of ENaC. Chimeric subunits were constructed between alpha and beta subunits (alpha-beta) and between gamma and beta subunits (gamma-beta). Chimeric and wild-type subunits were expressed in various combinations in Xenopus oocytes. Analysis of whole-cell and unitary currents made it possible to correlate functional properties with specific sequences in the subunits. Functional channels were generated without the second transmembrane domain from alpha subunits, indicating that it is not essential to form functional pores. The open probability and kinetics varied with the different channels and were influenced by the second hydrophobic domains. Amiloride affinity, Li(+)/Na(+) selectivity, and single channel conductance were also affected by this segment.     

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

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 (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. 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 alpha subunits separated by beta and gamma subunits.     

An Alu cassette in the human epithelial sodium channel

Here, we report the presence of two splice variants of the human epithelial sodium channel alpha subunit (h alpha ENaC) containing Alu cassette, namely h alpha ENaC+22 and h alpha ENaC+Alu, in various tissues. Functional expression of these splice variants with hENaC beta and gamma subunits produced loss-of-channel activity in the Xenopus oocyte expression system. Interestingly, coexpression of h alpha ENaC+22 or h alpha ENaC+Alu, respectively, with wild type hENaC alpha, beta, and gamma subunits enhanced the expression of amiloride-sensitive current in oocytes. The presence of Alu sequences in the 3'-untranslated region of h gamma ENaC was also identified.     

Characterization of the selectivity filter of the epithelial sodium channel

The epithelial sodium channel (ENaC) is composed of three homologous subunits termed alpha, beta, and gamma. Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the alpha-subunit to cysteine and co-expressing these mutants with wild type beta- and gamma-subunits of mouse ENaC in Xenopus laevis oocytes. Functional characteristics of these mutants were examined by two-electrode voltage clamp and single channel recording techniques. Two distinct domains were identified based on the functional changes associated with point mutations. An amino-terminal domain (alpha-Val(569)-alpha-Gly(579)) showed minimal changes in cation selectivity or amiloride sensitivity following cysteine substitution. In contrast, cysteine substitutions within the carboxyl-terminal domain (alpha-Ser(580)-alpha-Ser(592)) resulted in significant changes in cation selectivity and moderately altered amiloride sensitivity. The mutant channels containing alphaG587C or alphaS589C were permeable to K(+), and mutation of a GSS tract (positions alpha587-alpha589) to GYG resulted in a moderately K(+)-selective channel. Our results suggest that the C-terminal portion of the pore region within the alpha-subunit contributes to the selectivity filter of ENaC.     

Heterologous expression of a mammalian epithelial sodium channel in yeast

The alpha and beta subunits of the amiloride-sensitive rat epithelial sodium channel (alpha beta ENaC) were expressed in the yeast Saccharomyces cerevisiae. We used a combination of yeast strains, including a mutant in the secretory pathway (sec6), and Western blotting techniques, to show that alpha beta ENaC was synthesized and targeted through the secretory system to the plasma membrane. Yeasts expressing alpha beta ENaC were more sensitive to salt than the parent strain. In addition, amiloride, a specific blocker of ENaC, was found to suppress salt sensitivity in the yeast strain expressing alpha beta ENaC.     

Number of subunits comprising the epithelial sodium channel.

The human epithelial sodium channel (hENaC) is a hetero-oligomeric complex composed of three subunits, alpha, beta, and gamma. Understanding the structure and function of this channel and its abnormal behavior in disease requires knowledge of the number of subunits that comprise the channel complex. We used freeze-fracture electron microscopy and electrophysiological methods to evaluate the number of subunits in the ENaC complex expressed in Xenopus laevis oocytes. In oocytes expressing wild-type hENaC (alpha, beta, and gamma subunits), clusters of particles appeared in the protoplasmic face of the plasma membrane. The total number of particles in the clusters was consistent with the whole-cell amiloride-sensitive current measured in the same cells. The size frequency histogram for the particles in the clusters suggested the presence of an integral membrane protein complex composed of 17 +/- 2 transmembrane alpha-helices. Because each ENaC subunit has two putative transmembrane helices, these data suggest that in the oocyte plasma membrane, the ENaC complex is composed of eight or nine subunits. At high magnification, individual ENaC particles exhibited a near-square geometry. Functional studies using wild-type alphabeta-hENaC coexpressed with gamma-hENaC mutants, which rendered the functional channel differentially sensitive to methanethiosulfonate reagents and cadmium, suggested that the functional channel complex contains more than one gamma subunit. These data suggest that functional ENaC consists of eight or nine subunits of which a minimum of two are gamma subunits.     

Determination of epithelial Na+ channel subunit stoichiometry from single-channel conductances

The epithelial Na(+) channel (ENaC) is a multimeric membrane protein consisting of three subunits, alpha, beta, and gamma. The total number of subunits per functional channel complex has been described variously to follow either a tetrameric arrangement of 2alpha:1beta:1gamma or a higher-ordered stoichiometry of 3alpha:3beta:3gamma. Therefore, while it is clear that all three ENaC subunits are required for full channel activity, the number of the subunits required remains controversial. We used a new approach, based on single-channel measurements in Xenopus oocytes to address this issue. Individual mutations that alter single-channel conductance were made in pore-lining residues of ENaC alpha, beta, or gamma subunits. Recordings from patches in oocytes expressing a single species, wild type or mutant, of alpha, beta, and gamma showed a well-defined current transition amplitude with a single Gaussian distribution. When cRNAs for all three wild-type subunits were mixed with an equimolar amount of a mutant alpha-subunit (either S589D or S592T), amplitudes corresponding to pure wild-type or mutant conductances could be observed in the same patch, along with a third intermediate amplitude most likely arising from channels with at least one wild-type and at least 1 mutant alpha-subunit. However, intermediate or hybrid conductances were not observed with coexpression of wild-type and mutant betaG529A or gammaG534E subunits. Our results support a tetrameric arrangement of ENaC subunits where 2alpha, 1beta, and 1gamma come together around central pore.     

Association of the epithelial sodium channel with Apx and alpha-spectrin in A6 renal epithelial cells.

Recent molecular cloning of the epithelial sodium channel (ENaC) provides the opportunity to identify ENaC-associated proteins that function in regulating its cell surface expression and activity. We have examined whether ENaC is associated with Apx (apical protein Xenopus) and the spectrin-based membrane cytoskeleton in Xenopus A6 renal epithelial cells. We have also addressed whether Apx is required for the expression of amiloride-sensitive Na(+) currents by cloned ENaC. Sucrose density gradient centrifugation of A6 cell detergent extracts showed co-sedimentation of xENaC, alpha-spectrin, and Apx. Immunoblot analysis of proteins co-immunoprecipitating under high stringency conditions from peak Xenopus ENaC/Apx-containing gradient fractions indicate that ENaC, Apx, and alpha-spectrin are associated in a macromolecular complex. To examine whether Apx is required for the functional expression of ENaC, alphabetagamma mENaC cRNAs were coinjected into Xenopus oocytes with Apx sense or antisense oligodeoxynucleotides. The two-electrode voltage clamp technique showed there was a marked reduction in amiloride-sensitive current in oocytes coinjected with antisense oligonucleotides when to compared with oocytes coinjected with sense oligonucleotides. These studies indicate that ENaC is associated in a macromolecular complex with Apx and alpha-spectrin in A6 cells and suggest that Apx is required for the functional expression of ENaC in Xenopus epithelia.     

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.     

The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its alpha subunit

Epithelial sodium channels (ENaCs) mediate Na(+) entry across the apical membrane of high resistance epithelia that line the distal nephron, airway and alveoli, and distal colon. These channels are composed of three homologous subunits, termed alpha, beta, and gamma, which have intracellular amino and carboxyl termini and two membrane-spanning domains connected by large extracellular loops. Maturation of ENaC subunits involves furin-dependent cleavage of the extracellular loops at two sites within the alpha subunit and at a single site within the gamma subunit. The alpha subunits must be cleaved twice, immediately following Arg-205 and Arg-231, in order for channels to be fully active. Channels lacking alpha subunit cleavage are inactive with a very low open probability. In contrast, channels lacking both alpha subunit cleavage and the tract alphaAsp-206-Arg-231 are active when expressed in oocytes, suggesting that alphaAsp-206-Arg-231 functions as an inhibitor that stabilizes the channel in the closed conformation. A synthetic 26-mer peptide (alpha-26), corresponding to alphaAsp-206-Arg-231, reversibly inhibits wild-type mouse ENaCs expressed in Xenopus oocytes, as well as endogenous Na(+) channels expressed in either a mouse collecting duct cell line or primary cultures of human airway epithelial cells. The IC(50) for amiloride block of ENaC was not affected by the presence of alpha-26, indicating that alpha-26 does not bind to or interact with the amiloride binding site. Substitution of Arg residues within alpha-26 with Glu, or substitution of Pro residues with Ala, significantly reduced the efficacy of alpha-26. The peptide inhibits ENaC by reducing channel open probability. Our results suggest that proteolysis of the alpha subunit activates ENaC by disassociating an inhibitory domain (alphaAsp-206-Arg-231) from its effector site within the channel complex.     

Interaction of syntaxins with the amiloride-sensitive epithelial sodium channel.

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na(+) entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (alpha, beta, gamma subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.     

Small molecule activator of the human epithelial sodium channel

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of alpha, beta, and gamma subunits, belongs to the ENaC/degenerin family of ion channels and forms the principal route for apical Na(+) entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the beta subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of alphabetaG37Sgamma hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na(+) flux across epithelial membranes is clinically desirable.     

Endogenously expressed epithelial sodium channel is present in lipid rafts in A6 cells

The epithelial sodium channel (ENaC) present in the kidney collecting duct, distal colon, and the lung is responsible for salt reabsorption and whole body volume regulation. It is composed of three homologous subunits, alpha, beta, and gamma, and mutations to these subunits can lead to the salt wasting disease pseudohypoaldosteronism type I, associated with decreased channel density at the plasma membrane or to the hypertensive disorder, Liddle's syndrome, in which channel residency time at the plasma membrane is enhanced. Regulation of ENaC trafficking and turnover is therefore critical to sodium homeostasis. In this study we examined whether ENaC is present in the cholesterol-enriched microdomains commonly called lipid rafts, in the endogenously expressing A6 cell line. We demonstrate that a fraction of alpha, beta, and gamma ENaC is present in detergent-insoluble membranes, that subunits exist in membranes that float on discontinuous sucrose density gradients, and that methyl-beta-cyclodextrin treatment causes a redistribution of ENaC subunits to higher density membranes. Furthermore, chronic aldosterone stimulation results in a shift in the membrane density of all three subunits. Biotinylation of apical membrane proteins revealed that ENaC is present in lipid rafts on the plasma membrane. In conclusion, these results show that ENaC is present in lipid rafts both intracellularly and on the cell surface. Raft association may be important for trafficking and/or function of the channel.     

Functional heterogeneity of beta gamma-subunit of frog transducin

1. Transducin subunits (T alpha and T beta gamma) were purified from freshly dissected frog (Rana catesbeiana) retinas. It was found that purified T beta gamma is composed of three components which can be separated from each other by an anion exchange column chromatography under nondenaturing conditions. 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of these three components demonstrated that each contains T beta (mol. wt 35,000) and T gamma (mol. wt approximately 8000). 3. Only one of the three components retained an ability to enhance the binding of GppNHp to T alpha in the presence of a photobleaching intermediate of rhodopsin, while the others showed very low abilities to enhance the binding. 4. These observations, together with the similar findings on bovine T beta gamma, strongly suggest that the functional heterogeneity of T beta gamma is conserved in vertebrate photoreceptor cells.     

Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates epithelial sodium channel activity in A6 cells.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is a membrane lipid found in all eukaryotic cells, which regulates many important cellular processes, including ion channel activity. In this study, we used inside-out patch clamp technique, immunoprecipitation, and Western blot analysis to investigate the effect of PIP(2) on epithelial sodium channel activity in A6 cells. A6 cells were cultured in media supplemented with 1.5 microm aldosterone. Single sodium channel activity in excised, inside-out patches was increased by perfusion of the bath solution with 30 microm PIP(2) plus 100 microm GTP (NP(o) = 1.34 +/- 0.14) compared with the paired control (NP(o) = 0.09 +/- 0.02). However, neither 30 microm PIP(2) (NP(o) = 0.11 +/- 0.02) nor 100 microm GTP (NP(o) = 0.10 +/- 0.02) alone stimulated the sodium channels. The PIP(2)-stimulated channel activity was abolished by application of 10 nm G protein betagamma subunits (NP(o) = 0.14 +/- 0.05). However, 10 nm Galpha(i-3) + 30 microm PIP(2) increased both NP(o) and P(o). The stimulating effect of 10 nm Galpha(i-3) + 30 microm PIP(2) is similar to that of 30 microm PIP(2) plus 100 microm GTP. Immunoprecipitation and Western blot analysis show that both Gi(alpha-3) and PIP(2) bind beta and gamma epithelial Na(+) channels (ENaC), but not alpha ENaC. These results indicate that PIP(2) increases ENaC activity by direct interaction with beta or gamma xENaC in the presence of Galpha(i-3).     

Preferential assembly of epithelial sodium channel (ENaC) subunits in Xenopus oocytes: role of furin-mediated endogenous proteolysis

The epithelial sodium channel (ENaC) is preferentially assembled into heteromeric alphabetagamma complexes. The alpha and gamma (not beta) subunits undergo proteolytic cleavage by endogenous furin-like activity correlating with increased ENaC function. We identified full-length subunits and their fragments at the cell surface, as well as in the intracellular pool, for all homo- and heteromeric combinations (alpha, beta, gamma, alphabeta, alphagamma, betagamma, and alphabetagamma). We assayed corresponding channel function as amiloride-sensitive sodium transport (I(Na)). We varied furin-mediated proteolysis by mutating the P1 site in alpha and/or gamma subunit furin consensus cleavage sites (alpha(mut) and gamma(mut)). Our findings were as follows. (i) The beta subunit alone is not transported to the cell surface nor cleaved upon assembly with the alpha and/or gamma subunits. (ii) The alpha subunit alone (or in combination with beta and/or gamma) is efficiently transported to the cell surface; a surface-expressed 65-kDa alpha ENaC fragment is undetected in alpha(mut)betagamma, and I(Na) is decreased by 60%. (iii) The gamma subunit alone does not appear at the cell surface; gamma co-expressed with alpha reaches the surface but is not detectably cleaved; and gamma in alphabetagamma complexes appears mainly as a 76-kDa species in the surface pool. Although basal I(Na) of alphabetagamma(mut) was similar to alphabetagamma, gamma(mut) was not detectably cleaved at the cell surface. Thus, furin-mediated cleavage is not essential for participation of alpha and gamma in alphabetagamma heteromers. Basal I(Na) is reduced by preventing furin-mediated cleavage of the alpha, but not gamma, subunits. Residual current in the absence of furin-mediated proteolysis may be due to non-furin endogenous proteases.     

Fluorescence resonance energy transfer analysis of subunit stoichiometry of the epithelial Na+ channel

Activity of the epithelial Na(+) channel (ENaC) is rate-limiting for Na(+) (re)absorption across electrically tight epithelia. ENaC is a heteromeric channel comprised of three subunits, alpha, beta, and gamma, with each subunit contributing to the functional channel pore. The subunit stoichiometry of ENaC remains uncertain with electrophysiology and biochemical experiments supporting both a tetramer with a 2alpha:1beta:1gamma stoichiometry and a higher ordered channel with a 3alpha:3beta:3gamma stoichiometry. Here we used an independent biophysical approach based upon fluorescence resonance energy transfer (FRET) between differentially fluorophore-tagged ENaC subunits to determine the subunit composition of mouse ENaC functionally reconstituted in Chinese hamster ovary and COS-7 cells. We found that when all three subunits were co-expressed, ENaC contained at least two of each type of subunit. Findings showing that ENaC subunits interact with similar subunits in immunoprecipitation studies are consistent with these FRET results. Upon native polyacrylamide gel electrophoresis, moreover, oligomerized ENaC runs predominantly as a single species with a molecular mass of >600 kDa. Because single ENaC subunits have a molecular mass of approximately 90 kDa, these results also agree with the FRET results. The current results as a whole, thus, are most consistent with a higher ordered channel possibly with a 3alpha:3beta:3gamma stoichiometry.     

Distinct pools of epithelial sodium channels are expressed at the plasma membrane

The epithelial Na+ channel (ENaC) is assembled in the endoplasmic reticulum from three structurally related subunits (alpha, beta, and gamma). Channel maturation within the biosynthetic pathway involves cleavage of the alpha and gamma subunits by furin and processing of N-linked glycans on alpha, beta, and gamma to complex type. Both mature and immature subunits have been observed at the surface of stably transfected Madin-Darby canine kidney cells. We have examined whether channel maturation is an all-or-none event or whether heterogeneous processing of channel subunits occurs within an individual channel complex. Using an immobilized lectin to isolate proteins with complex type N-glycans, we found that individual channel complexes with mature subunits lack immature subunits. Furthermore, terminal processing of N-glycans on ENaC subunits was not dependent on cleavage of ENaC subunits, and proteolysis of channel subunits was not dependent on prior processing of N-glycans. Our results suggest that processing of subunits within an individual channel complex is an all-or-none event such that channels present on the cell surface contain either all mature or all immature subunits. The presence of immature channel complexes at the plasma membrane provides epithelial cells with a reserve of poorly functional channels that can be activated by proteases in post-Golgi compartments.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.