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+.     

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.     

Paradoxical stimulation of a DEG/ENaC channel by amiloride.

Extracellular amiloride inhibits all known DEG/ENaC ion channels, including BNC1, a proton-activated human neuronal cation channel. Earlier studies showed that protons cause a conformational change that activates BNC1 and exposes residue 430 to the extracellular solution. Here we demonstrate that, in addition to blocking BNC1, amiloride also exposes residue 430. This result suggested that, like protons, amiloride might be capable of activating the channel. To test this hypothesis, we introduced a mutation in the BNC1 pore that reduces amiloride block, and found that amiloride stimulated these channels. Amiloride inhibition was voltage-dependent, suggesting block within the pore, whereas stimulation was not, suggesting binding to an extracellular site. These data show that amiloride can have two distinct effects on BNC1, and they suggest two different interaction sites. The results suggest that extracellular amiloride binding may have a stimulatory effect similar to that of protons in BNC1 or extracellular ligands in other DEG/ENaC channels.     

Gain-of-function mutations in the MEC-4 DEG/ENaC sensory mechanotransduction channel alter gating and drug blockade

MEC-4 and MEC-10 are the pore-forming subunits of the sensory mechanotransduction complex that mediates touch sensation in Caenorhabditis elegans (O'Hagan, R., M. Chalfie, and M.B. Goodman. 2005. Nat. Neurosci. 8:43-50). They are members of a large family of ion channel proteins, collectively termed DEG/ENaCs, which are expressed in epithelial cells and neurons. In Xenopus oocytes, MEC-4 can assemble into homomeric channels and coassemble with MEC-10 into heteromeric channels (Goodman, M.B., G.G. Ernstrom, D.S. Chelur, R. O'Hagan, C.A. Yao, and M. Chalfie. 2002. Nature. 415:1039-1042). To gain insight into the structure-function principles that govern gating and drug block, we analyzed the effect of gain-of-function mutations using a combination of two-electrode voltage clamp, single-channel recording, and outside-out macropatches. We found that mutation of A713, the d or degeneration position, to residues larger than cysteine increased macroscopic current, open probability, and open times in homomeric channels, suggesting that bulky residues at this position stabilize open states. Wild-type MEC-10 partially suppressed the effect of such mutations on macroscopic current, suggesting that subunit-subunit interactions regulate open probability. Additional support for this idea is derived from an analysis of macroscopic currents carried by single-mutant and double-mutant heteromeric channels. We also examined blockade by the diuretic amiloride and two related compounds. We found that mutation of A713 to threonine, glycine, or aspartate decreased the affinity of homomeric channels for amiloride. Unlike the increase in open probability, this effect was not related to size of the amino acid side chain, indicating that mutation at this site alters antagonist binding by an independent mechanism. Finally, we present evidence that amiloride block is diffusion limited in DEG/ENaC channels, suggesting that variations in amiloride affinity result from variations in binding energy as opposed to accessibility. We conclude that the d position is part of a key region in the channel functionally and structurally, possibly representing the beginning of a pore-forming domain.     

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.     

Mutations in the pore region modify epithelial sodium channel gating by shear stress

Previous studies have shown that epithelial Na+ channels (ENaCs) are activated by laminar shear stress (LSS). ENaCs with a high intrinsic open probability because of a mutation (betaS518K) or covalent modification of an introduced Cys residue (alphaS580C) in the pre-second transmembrane domain (pre-M2) were not activated by LSS, suggesting that the pre-M2 region participates in conformational rearrangements during channel activation. We examined the role of the pore region of the alpha-subunit in channel gating by studying the kinetics of activation by LSS of wild-type ENaC and channels with Cys mutations in the tract Ser576-Ser592. Whole cell Na+ currents were monitored in oocytes expressing wild-type or mutant ENaCs prior to and following application of LSS. Following a 2.2-s delay, a monoexponential increase in Na+ currents was observed with a time constant (tau) of 8.1 s in oocytes expressing wild-type ENaC. Cys substitutions within the alpha-subunit in the tract Ser580-Ser589 resulted in: (i) a reduction (Ser580-Trp585, Gly587) or increase (Ser589) in delay times preceding channel activation by LSS, (ii) an increase (Gln581, Leu584, Trp585, Phe586, Ser588) or decrease (Ser589) in the rate of channel activation, or (iii) a decrease in the magnitude of the response (Ser583, Gly587, Leu584). Cys substitutions at a putative amiloride-binding site (alphaSer583 or betaGly525) or within the selectivity filter (alphaGly587) resulted in a reduction in the LSS response, and exhibited a multiexponential time course of activation. The corresponding gamma-subunit mutant (alphabetagammaG542C) had a minimal response to LSS and exhibited a high intrinsic open probability. These data suggest that residues in the pore region participate in the sensing and/or transduction of the mechanical stimulus that results in channel activation and are consistent with the hypothesis that the ENaC pore region has a key role in modulating channel gating.     

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.     

Interactions of amiloride and other blocking cations with the apical Na channel in the toad urinary bladder

Summary A simple model of the action of amiloride to block apical Na channels in the toad urinary bladder was tested. According to the model, the positively charged form of the drug binds to a site in the lumen of the channel within the electric field of the membrane. In agreement with the predictions of the model: (1) The voltage dependence of amiloride block was consistent with the assumption of a single amiloride binding site, at which about 15% of the transmembrane voltage is sensed, over a voltage range of ±160 mV. (2) The time course of the development of voltage dependence was consistent with that predicted from the rate constants for amiloride binding previously determined. (3) The ability of organic cations to mimic the action of amiloride showed a size dependence implying a restriction of access to the binding site, with an effective diameter of about 5 angstroms. In a fourth test, divalent cations (Ca, Mg, Ba and Sr) were found to block Na channels with a complex voltage dependence, suggesting that these ions interact with two or more sites. at least one of which may be within the lumen of the pore.     

Asymmetric organization of the pore region of the epithelial sodium channel

Epithelial sodium channels (ENaCs) are composed of three homologous subunits that have regions preceding the second transmembrane domain (also referred as pre-M2) that form part of the channel pore. To identify residues within this region of the beta-subunit that line the pore, we systematically mutated residues Gln(523)-Ile(536) to cysteine. Wild type and mutant mouse ENaCs were expressed in Xenopus oocytes, and a two-electrode voltage clamp was used to examine the properties of mutant channels. Cysteine substitutions of 9 of 13 residues significantly altered Li(+) to Na(+) current ratios, whereas only cysteine replacement of beta Gly(529) resulted in K(+)-permeable channels. Besides beta G525C, large increases in the inhibitory constant of amiloride were observed with mutations at beta Gly(529) and beta Ser(531) within the previously identified 3-residue tract that restricts K(+) permeation. Cysteine substitution preceding (beta Phe(524) and beta Gly(525)), within (beta Gly(530)) or following (beta Leu(533)) this 3-residue tract, resulted in enhanced current inhibition by external MTSEA. External MTSET partially blocked channels with cysteine substitutions at beta Gln(523), beta Phe(524), and beta Trp(527). MTSET did not inhibit alpha beta G525C gamma, although previous studies showed that channels with cysteine substitutions at the corresponding sites within the alpha- and gamma-subunits were blocked by MTSET. Our results, placed in context with previous observations, suggest that pore regions from the three ENaC subunits have an asymmetric organization.     

Identification of a functional phosphatidylinositol 3,4,5-trisphosphate binding site in the epithelial Na+ channel

Membrane phospholipids, such as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)), are signaling molecules that can directly modulate the activity of ion channels, including the epithelial Na(+) channel (ENaC). Whereas PI(3,4,5)P(3) directly activates ENaC, its binding site within the channel has not been identified. We identify here a region of gamma-mENaC just following the second trans-membrane domain (residues 569-583) important to PI(3,4,5)P(3) binding and regulation. Deletion of this track decreases activity of ENaC heterologously expressed in Chinese hamster ovary cells. K-Ras and its first effector phosphoinositide 3-OH kinase (PI3-K), as well as RhoA and its effector phosphatidylinositol 4-phosphate 5-kinase increase ENaC activity. Whereas the former, via generation of PI(3,4,5)P(3), increases ENaC open probability, the latter increases activity by increasing membrane levels of the channel. Deletion of the region just distal to the second trans-membrane domain disrupted regulation by K-Ras and PI3-K but not RhoA and phosphatidylinositol 4-phosphate 5-kinase. Moreover, PI(3,4,5)P(3) binds ENaC with deletion of the region following the second transmembrane domain disrupting this interaction and disrupting direct activation of the channel by PI(3,4,5)P(3). Mutation analysis revealed the importance of conserved positive and negative charged residues as well as bulky amino acids within this region to modulation of ENaC by PI3-K. The current results identify the region just distal to the second trans-membrane domain within gamma-mENaC as being part of a functional PI(3,4,5)P(3) binding site that directly impacts ENaC activity. Phospholipid binding to this site is probably mediated by the positively charged amino acids within this track, with negatively charged and bulky residues also influencing specificity of interactions.     

Antiidiotypic antibody recognizes an amiloride binding domain within the alpha subunit of the epithelial Na+ channel

We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na+ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na+ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis alphaENaC. RA6.3 specifically recognized an amiloride binding domain within the alpha-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the alpha-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to alphaENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within alphaENaC.     

Gating induces a conformational change in the outer vestibule of ENaC

The epithelial Na(+) channel (ENaC) is comprised of three homologous subunits (alpha, beta, and gamma). The channel forms the pathway for Na(+) absorption in the kidney, and mutations cause disorders of Na(+) homeostasis. However, little is known about the mechanisms that control the gating of ENaC. We investigated the gating mechanism by introducing bulky side chains at a position adjacent to the extracellular end of the second membrane spanning segment (549, 520, and 529 in alpha, beta, and gammaENaC, respectively). Equivalent "DEG" mutations in related DEG/ENaC channels in Caenorhabditis elegans cause swelling neurodegeneration, presumably by increasing channel activity. We found that the Na(+) current was increased by mutagenesis or chemical modification of this residue and adjacent residues in alpha, beta, and gammaENaC. This resulted from a change in the gating of ENaC; modification of a cysteine at position 520 in betaENaC increased the open state probability from 0. 12 to 0.96. Accessibility to this side chain from the extracellular side was state-dependent; modification occurred only when the channel was in the open conformation. Single-channel conductance decreased when the side chain contained a positive, but not a negative charge. However, alterations in the side chain did not alter the selectivity of ENaC. This is consistent with a location for the DEG residue in the outer vestibule. The results suggest that channel gating involves a conformational change in the outer vestibule of ENaC. Disruption of this mechanism could be important clinically since one of the mutations that increased Na(+) current (gamma(N530K)) was identified in a patient with renal disease.     

Regulation of epithelial sodium channel activity through a region of the carboxyl terminus of the alpha -subunit. Evidence for intracellular kinase-mediated reactions

The epithelial sodium channel (ENaC) is a heteromultimer composed of three subunits, each having two membrane-spanning domains with intracellular amino and carboxyl termini. Several hormones and proteins regulate channel activity, but the molecular nature of this regulation is unknown. We conducted experiments to determine a possible new site within the carboxyl terminus of the alpha-subunit involved in enhanced channel activity through endogenous kinases. When an alpha-subunit that was truncated to remove a PY motif was expressed in Xenopus oocytes with wild type human beta- and gamma-ENaC subunits, channel activity was greatly enhanced. The removal of the entire intracellular carboxyl terminus of the alpha-subunit eliminated this enhanced basal activity. Using several point mutations, we localized this site to two amino acid residues (Pro(595)-Gly(596)) near the second membrane-spanning domain. The nonspecific kinase inhibitor staurosporine inhibits basal channel activity of wild type ENaC but was ineffective in inhibiting channels mutated at this site. The major effect of these mutations was not on channel kinetics but was largely, if not entirely, on the number of active channels on the cell surface. This region is potentially important in effecting kinase-mediated increases in ENaC activity.     

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.     

Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

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.     

Regulation of stability and function of the epithelial Na+ channel (ENaC) by ubiquitination.

The epithelial Na+ channel (ENaC), composed of three subunits (alpha beta gamma), plays a critical role in salt and fluid homeostasis. Abnormalities in channel opening and numbers have been linked to several genetic disorders, including cystic fibrosis, pseudohypoaldosteronism type I and Liddle syndrome. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein of ENaC. Here we show that ENaC is a short-lived protein (t1/2 approximately 1 h) that is ubiquitinated in vivo on the alpha and gamma (but not beta) subunits. Mutation of a cluster of Lys residues (to Arg) at the N-terminus of gamma ENaC leads to both inhibition of ubiquitination and increased channel activity, an effect augmented by N-terminal Lys to Arg mutations in alpha ENaC, but not in beta ENaC. This elevated channel activity is caused by an increase in the number of channels present at the plasma membrane; it represents increases in both cell-surface retention or recycling of ENaC and incorporation of new channels at the plasma membrane, as determined by Brefeldin A treatment. In addition, we find that the rapid turnover of the total pool of cellular ENaC is attenuated by inhibitors of both the proteasome and the lysosomal/endosomal degradation systems, and propose that whereas the unassembled subunits are degraded by the proteasome, the assembled alpha beta gamma ENaC complex is targeted for lysosomal degradation. Our results suggest that ENaC function is regulated by ubiquitination, and propose a paradigm for ubiquitination-mediated regulation of ion channels.     

Distinct structural elements in the first membrane-spanning segment of the epithelial sodium channel

Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an alpha2betagamma or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate site within alphaM1. We performed site-directed mutagenesis at this site and found that, although the mutants reduced channel activity, ENaC protein expression at the plasma membrane was unaffected. To deduce the role of alphaM1 in the pore structure of ENaC, we performed tryptophan-scanning mutagenesis throughout alphaM1 (residues 110-130). We found that mutations within the amino-terminal part of alphaM1 had effects on activity and selectivity with a periodicity consistent with a helical structure but no effect on channel surface expression. We also observed that mutations within the carboxyl-terminal part of alphaM1 had effects on activity and selectivity but with no apparent periodicity. Additionally, these mutants reduced channel surface expression. Our data support a model in which the amino-terminal half of alphaM1 is alpha-helical and packs against structural element(s) that contribute to the ENaC pore. Furthermore, these data suggest that the carboxyl-terminal half of alphaM1 may be helical or assume a different conformation and may be involved in tertiary interactions essential to proper channel folding or assembly. Together, our data suggest that alphaM1 is divided into two distinct regions.     

Epithelial sodium channel pore region. structure and role in gating

Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.     

Discovery of a novel chemotype of potent human ENaC blockers using a bioisostere approach. Part 2: α-Branched quaternary amines

We report the synthesis and biological evaluation of a series of novel α-branched pyrazinoyl quaternary amines for their ability to block ion transport via the epithelial sodium channel (ENaC) in human bronchial epithelial cells (HBECs). Compound 12 g has an IC(50) of 30 nM and is highly efficacious in the Guinea-pig tracheal potential difference (TPD) model of ENaC blockade with an ED(50) of 1 μg kg(-1) at 1h. In addition the SAR results demonstrate for the first time the chiral nature of the binding site of human ENaC. As such, pyrazinoyl quaternary amines represent a promising new class of ENaC blockers for the treatment of cystic fibrosis that are structurally distinct from the pyrazinoyl guanidine chemotype found in prototypical ENaC blockers such as amiloride.