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Probing the Structural Basis of Zn2+ Regulation of the Epithelial Na+ Channel*

Open AccessPublished:August 28, 2012DOI:https://doi.org/10.1074/jbc.M112.394734
      Extracellular Zn2+ activates the epithelial Na+ channel (ENaC) by relieving Na+ self-inhibition. However, a biphasic Zn2+ dose response was observed, suggesting that Zn2+ has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn2+, we examined the effects of mutating the 10 extracellular His residues of mouse γENaC. Four mutations within the finger subdomain (γH193A, γH200A, γH202A, and γH239A) significantly reduced the maximal Zn2+ activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn2+ activating site, γH239A diminished Na+ self-inhibition and thus concealed the activating effects of Zn2+. Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn2+ inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp516 was predicted to be in close proximity to γHis88. Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn2+. Our results suggest that external Zn2+ regulates ENaC activity by binding to multiple extracellular sites within the γ-subunit, including (i) a high-affinity stimulatory site within the finger subdomain involving His193, His200, and His202 and (ii) a low-affinity Zn2+ inhibitory site within the palm subdomain that includes His88 and Asp516.

      Introduction

      Sodium transport mediated by the epithelial Na+ channel (ENaC)
      The abbreviations used are: ENaC
      epithelial Na+ channel
      γmENaC
      mouse γENaC
      cASIC1
      chicken acid-sensing ion channel 1
      ECD
      extracellular domain.
      plays an important role in the maintenance of extracellular fluid volume and regulation of blood pressure and in the regulation of airway surface liquid volume. Altered channel activity induced by mutations of ENaC genes results in several disorders, including Liddle syndrome and pseudohypoaldosteronism type 1, and contributes to the pathogenesis of cystic fibrosis (
      • Rossier B.C.
      • Pradervand S.
      • Schild L.
      • Hummler E.
      Epithelial sodium channel and the control of sodium balance: interaction between genetic and environmental factors.
      ,
      • Snyder P.M.
      The epithelial Na+ channel: cell surface insertion and retrieval in Na+ homeostasis and hypertension.
      ,
      • Bhalla V.
      • Hallows K.R.
      Mechanisms of ENaC regulation and clinical implications.
      ,
      • Qadri Y.J.
      • Rooj A.K.
      • Fuller C.M.
      ENaCs and ASICs as therapeutic targets.
      ). Specific ENaC variants are suggested to contribute to the genesis of essential hypertension in selected populations (
      • Pratt J.H.
      Central role for ENaC in development of hypertension.
      ,
      • Soundararajan R.
      • Pearce D.
      • Hughey R.P.
      • Kleyman T.R.
      Role of epithelial sodium channels and their regulators in hypertension.
      ,
      • Büsst C.J.
      • Bloomer L.D.
      • Scurrah K.J.
      • Ellis J.A.
      • Barnes T.A.
      • Charchar F.J.
      • Braund P.
      • Hopkins P.N.
      • Samani N.J.
      • Hunt S.C.
      • Tomaszewski M.
      • Harrap S.B.
      The epithelial sodium channel γ-subunit gene and blood pressure: family-based association, renal gene expression, and physiological analyses.
      ).
      ENaC activity is regulated by a variety of extracellular factors, including proteases, shear stress, pH, anions, nucleotides, and transition metals (
      • Van Driessche W.
      • Zeiske W.
      Ionic channels in epithelial cell membranes.
      ,
      • Sheng S.
      • Johnson J.P.
      • Kleyman T.R.
      ,
      • Yu L.
      • Eaton D.C.
      • Helms M.N.
      Effect of divalent heavy metals on epithelial Na+ channels in A6 cells.
      ,
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
      ,
      • Collier D.M.
      • Snyder P.M.
      Extracellular protons regulate human ENaC by modulating Na+ self-inhibition.
      ,
      • Collier D.M.
      • Snyder P.M.
      Extracellular chloride regulates the epithelial sodium channel.
      ,
      • Chen J.
      • Myerburg M.M.
      • Passero C.J.
      • Winarski K.L.
      • Sheng S.
      External Cu2+ inhibits human epithelial Na+ channels by binding at a subunit interface of extracellular domains.
      ,
      • Molina R.
      • Han D.Y.
      • Su X.F.
      • Zhao R.Z.
      • Zhao M.
      • Sharp G.M.
      • Chang Y.
      • Ji H.L.
      Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition.
      ). Although some metals are essential for normal physiological function, excessive environmental exposure can be toxic (
      • Mathie A.
      • Sutton G.L.
      • Clarke C.E.
      • Veale E.L.
      Zinc and copper: pharmacological probes and endogenous modulators of neuronal excitability.
      ,
      • St Croix C.M.
      • Leelavaninchkul K.
      • Watkins S.C.
      • Kagan V.E.
      • Pitt B.R.
      Nitric oxide and zinc homeostasis in acute lung injury.
      ). For instance, particulate matter and airborne particles containing high amounts of transitional metals, including zinc and copper, contribute to pulmonary and cardiovascular toxicity (
      • Adamson I.Y.
      • Prieditis H.
      • Vincent R.
      Pulmonary toxicity of an atmospheric particulate sample is due to the soluble fraction.
      ,
      • Prieditis H.
      • Adamson I.Y.
      Comparative pulmonary toxicity of various soluble metals found in urban particulate dusts.
      ). Although recent studies have suggested that ion channels are potential molecular targets of transition metals (
      • Restrepo-Angulo I.
      • De Vizcaya-Ruiz A.
      • Camacho J.
      Ion channels in toxicology.
      ), the mechanisms that confer the harmful effects of heavy metals are poorly understood.
      Zinc is the second most abundant transition metal in living organisms and is thought to complex with ∼10% of the human proteome (
      • Andreini C.
      • Banci L.
      • Bertini I.
      • Rosato A.
      Counting the zinc proteins encoded in the human genome.
      ). It has catalytic, structural, or regulatory roles in proteins that are involved in diverse biological processes (
      • Chasapis C.T.
      • Loutsidou A.C.
      • Spiliopoulou C.A.
      • Stefanidou M.E.
      Zinc and human health: an update.
      ). For example, zinc regulates voltage- and ligand-gated ion channels and may function as a signaling ion in brain (
      • Harrison N.L.
      • Gibbons S.J.
      Zn2+: an endogenous modulator of ligand- and voltage-gated ion channels.
      ,
      • Frederickson C.J.
      • Koh J.Y.
      • Bush A.I.
      The neurobiology of zinc in health and disease.
      ). Zinc may play a pathophysiological role in several disorders such as Alzheimer disease, cancer, diabetes, and depression (
      • Chasapis C.T.
      • Loutsidou A.C.
      • Spiliopoulou C.A.
      • Stefanidou M.E.
      Zinc and human health: an update.
      ).
      Previous studies have demonstrated that low concentrations of extracellular Zn2+ activate ENaC. Zn2+ increases short-circuit current in amphibian epithelia expressing native Na+ channels (
      • Van Driessche W.
      • Zeiske W.
      Ionic channels in epithelial cell membranes.
      ). Zn2+ activates ENaCs in heterologous expression systems by directly interacting with the channel and altering channel gating (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition.
      ). Single channel recordings revealed that external Zn2+ increased the number of observed channels within a patch without changing single channel conductance (
      • Yu L.
      • Eaton D.C.
      • Helms M.N.
      Effect of divalent heavy metals on epithelial Na+ channels in A6 cells.
      ). The response of ENaC to extracellular Zn2+ is biphasic. Low concentrations of Zn2+ activate the channel, with a maximal response at 100 μm Zn2+. Further increases in [Zn2+] reduce channel activity. This bell-shaped dose-response relationship suggests that Zn2+ enhances channel activity at low concentrations and is inhibitory at high concentrations (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition.
      ). The stimulatory effect of Zn2+ on ENaC has been attributed to relief of Na+ self-inhibition (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition.
      ). Na+ self-inhibition is a rapid down-regulation of ENaC open probability when the extracellular Na+ concentration increases (
      • Sheng S.
      • Johnson J.P.
      • Kleyman T.R.
      ,
      • Kashlan O.B.
      • Kleyman T.R.
      ENaC structure and function in the wake of a resolved structure of a family member.
      ). However, the Zn2+-binding sites within ENaC have not been identified, and the biphasic dose response to the heavy metal has not been explained.
      ENaCs are typically composed of three homologous subunits, each of which contains short intracellular N and C termini, two transmembrane domains, and a large extracellular region. The crystal structure of the chicken acid-sensing ion channel 1 (cASIC1; homologous to ENaC) reveals a trimeric channel complex with a large extracellular domain (ECD) connected to the transmembrane domain via a wrist region (
      • Jasti J.
      • Furukawa H.
      • Gonzales E.B.
      • Gouaux E.
      Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH.
      ). The extracellular region of a subunit is organized into five subdomains, referred to as palm, β-ball, knuckle, finger, and thumb. Recent studies suggest that the extracellular regions of ENaC subunits have a similar overall design to that of cASIC1 and that the ECD may sense various extracellular signals (
      • Collier D.M.
      • Snyder P.M.
      Extracellular protons regulate human ENaC by modulating Na+ self-inhibition.
      ,
      • Chen J.
      • Myerburg M.M.
      • Passero C.J.
      • Winarski K.L.
      • Sheng S.
      External Cu2+ inhibits human epithelial Na+ channels by binding at a subunit interface of extracellular domains.
      ,
      • Collier D.M.
      • Snyder P.M.
      Identification of epithelial Na+ channel (ENaC) intersubunit Cl inhibitory residues suggests a trimeric αγβ channel architecture.
      ,
      • Shi S.
      • Ghosh D.D.
      • Okumura S.
      • Carattino M.D.
      • Kashlan O.B.
      • Sheng S.
      • Kleyman T.R.
      Base of the thumb domain modulates epithelial sodium channel gating.
      ,
      • Kashlan O.B.
      • Adelman J.L.
      • Okumura S.
      • Blobner B.M.
      • Zuzek Z.
      • Hughey R.P.
      • Kleyman T.R.
      • Grabe M.
      Constraint-based homology model of the extracellular domain of the epithelial Na+ channel α-subunit reveals a mechanism of channel activation by proteases.
      ,
      • Stewart A.P.
      • Haerteis S.
      • Diakov A.
      • Korbmacher C.
      • Edwardson J.M.
      Atomic force microscopy reveals the architecture of the epithelial sodium channel (ENaC).
      ). However, the exact locations where most extracellular factors interact with ENaC and the mechanistic details of how the initial contacts lead to altered channel activity remain elusive.
      The ECDs of ENaC subunits contain numerous residues, such as His, Cys, Glu, and Asp, which are capable of serving as Zn2+-binding ligands (
      • Dokmanić I.
      • Sikić M.
      • Tomić S.
      Metals in proteins: correlation between the metal-ion type, coordination number, and the amino acid residues involved in the coordination.
      ). We hypothesized that some of these residues mediate Zn2+ interaction with ENaC. Several lines of evidence suggest that the γ-subunit has a particularly important role in regulating channel gating in response to external factors (
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
      ,
      • Carattino M.D.
      • Hughey R.P.
      • Kleyman T.R.
      Proteolytic processing of the epithelial sodium channel γ-subunit has a dominant role in channel activation.
      ,
      • Diakov A.
      • Bera K.
      • Mokrushina M.
      • Krueger B.
      • Korbmacher C.
      Cleavage in the γ-subunit of the epithelial sodium channel (ENaC) plays an important role in the proteolytic activation of near-silent channels.
      ). Mutations were introduced into His residues within the γ-subunit ECD to identify sites that have a role in the regulation of ENaC by Zn2+. Three His residues were identified within the finger subdomain that participate in a high-affinity Zn2+ activating site. Additionally, mutation of a palm subdomain His residue altered the low-affinity inhibitory response. These His residues are unique to the γ-subunit, as they are not found at the homologous sites in the α- and β-subunits.

      DISCUSSION

      The major goal of this study was to probe the structural basis of the regulation of ENaC by Zn2+. The biphasic dose response of Zn2+ on the purinergic receptor P2X is believed to originate from Zn2+ binding at two distinct sites within the receptor: a high-affinity activating site and a low-affinity inhibitory site (
      • Coddou C.
      • Yan Z.
      • Obsil T.
      • Huidobro-Toro J.P.
      • Stojilkovic S.S.
      Activation and regulation of purinergic P2X receptor channels.
      ). Because the ENaC/degenerin family is structurally similar to P2X (
      • Gonzales E.B.
      • Kawate T.
      • Gouaux E.
      Pore architecture and ion sites in acid-sensing ion channels and P2X receptors.
      ), we hypothesized that the bell-shaped Zn2+ dose-response relationship with ENaC activity is due to the presence of a high-affinity activating site and a low-affinity inhibitory site within the ECD. We found that a minimal two-site model sufficiently describes the bell-shaped dose response of mouse ENaC to external Zn2+. The presence of a high-affinity activating site and a low-affinity inhibitory site is supported by our observations that Zn2+-mediated activation or inhibition can be selectively eliminated by targeted mutations of specific residues in the activating site or the inhibitory site, respectively. On the basis of these observations, we propose that γHis193, γHis200, and γHis202 within the finger subdomain contribute to a high-affinity Zn2+-binding site and that γHis88 and γAsp516 in the palm subdomain are key determinants of a low-affinity Zn2+-binding site (Fig. 9).
      Figure thumbnail gr9
      FIGURE 9Finger and palm subdomains of γENaC contain key sites for Zn2+ interaction with the channel. A, a structural model of cASIC1. Three ASIC1 subunits are rendered as colored ribbons from coordinates in the Protein Data Bank (code 3HGC) (
      • Gonzales E.B.
      • Kawate T.
      • Gouaux E.
      Pore architecture and ion sites in acid-sensing ion channels and P2X receptors.
      ). The red circle identifies the finger subdomain of ASIC1 to show the likely locations of γHis193, γHis200, and γHis202 that are proposed to contribute to the high-affinity activating Zn2+-binding site. Their exact homologous sites within the ASIC1 structure are not known due to a low level of sequence homology in the finger subdomains. The sequence alignments of mouse, rat, and human α-, β-, and γ-subunits and cASIC1 were performed with Vector NTI 11.0 (Invitrogen). Rat and human ENaC sequences are omitted for clarity. The three His residues are shown in red. The red square shows a subunit interface between the palm subdomain of the green subunit and the thumb subdomain of the blue subunit, where γHis88 and γAsp516 are proposed to reside in a mENaC structure and contribute to the low-affinity inhibitory Zn2+-binding site. The green ASIC1 subunit was arbitrarily considered to correspond to γmENaC. The sequences of loops β1-β2 and β11-β12 of αβγmENaC and cASIC1 are shown with identical residues highlighted in yellow and γHis88 and γAsp516 in red. B, enlargement of the area enclosed by the red square in A. Three β-strands (β1, β11, and β12) are labeled as in the literature (
      • Gonzales E.B.
      • Kawate T.
      • Gouaux E.
      Pore architecture and ion sites in acid-sensing ion channels and P2X receptors.
      ). The γHis88-homologous Ala82 and γAsp516-homologous Ala413 residues were mutated to His and Asp, respectively, in the ASIC1 model. The labeled blue sphere shows Cl bound within the thumb subdomain of the blue subunit. Structural models were drawn using PyMOL 1.3 (
      • DeLano W.L.
      ).
      The following observations suggest that γHis193, γHis200, and γHis202, presumably within the finger subdomain, contribute to a high-affinity Zn2+ activating site. (i) Mutation of these residues increased the EC50 of Zn2+ activation and had no effect on the IC50 (Fig. 5 and Table 1), suggesting that these residues are specifically involved in Zn2+ binding to an activating site. (ii) Although mutations of γHis193, γHis200, and γHis202 reduced the maximal ENaC current elicited by Zn2+, maximal Zn2+ activation was not affected when the Zn2+ inhibitory site was also mutated (γH88A/γH200A/γH202A). Thus, γH200A/γH202A appears to primarily affect the apparent affinity of the activating site and does not alter the efficacy of channel activation. (iii) γHis193, γHis200, and γHis202 appear to share a similar functional role in mediating Zn2+ effects on the channel. Individual mutation of any of the three His residues induced a similar phenotype, including an increase in EC50, a decrease in maximal Zn2+ activation, and a lack of effect on Na+ self-inhibition. Moreover, mutation-induced changes in EC50 were not additive in γH193A/γH200A/γH202A channels (Fig. 6D). (iv) Their proximity in the sequence makes it structurally possible for them to contribute to a common binding site. Interestingly, the His200-Val201-His202 sequence matches a common Zn2+-binding motif, HXH, where X can be any residue (
      • Karlin S.
      • Zhu Z.Y.
      Classification of mononuclear zinc metal sites in protein structures.
      ). This motif is also present in other channels (
      • Choi Y.B.
      • Lipton S.A.
      Identification and mechanism of action of two histidine residues underlying high-affinity Zn2+ inhibition of the NMDA receptor.
      ,
      • Harvey R.J.
      • Thomas P.
      • James C.H.
      • Wilderspin A.
      • Smart T.G.
      Identification of an inhibitory Zn2+-binding site on the human glycine receptor α1-subunit.
      ).
      Channels with substitutions of all three His residues are still activated by Zn2+, suggesting that there are additional sites within the channel where Zn2+ binds and activates ENaC. It is notable that His residues are not present at sites in the α- and β-subunits that correspond to γHis193, γHis200, and γHis202 (Fig. 9). Further studies are needed to identify additional sites involved in Zn2+ activation of ENaC.
      Our results demonstrate that γHis88 within the palm subdomain is a key determinant for the low-affinity inhibitory Zn2+-binding site. Interestingly, like the three finger subdomain His residues, γHis88 is also not conserved among ENaC subunits (Fig. 9). Taking advantage of the structural information of ASIC1, a negatively charged residue, γAsp516, was identified that lies in close proximity to γHis88 and is likely involved in the inhibitory effect of Zn2+ as well (Fig. 9). However, it is also possible that mutations of γHis88 and γAsp516 may diminish the inhibitory component in the biphasic Zn2+ dose response by disrupting conformational changes induced by Zn2+ binding, instead of abolishing Zn2+ binding. Nevertheless, our observations suggest that this region (loops β1-β2 and β11-β12), located at the middle of the palm subdomain, contributes to a general inhibitory site for different channel regulators (Fig. 9). Indeed, a putative Cl-binding site and a Cu2+-binding site have been identified near this region in human ENaCs (
      • Chen J.
      • Myerburg M.M.
      • Passero C.J.
      • Winarski K.L.
      • Sheng S.
      External Cu2+ inhibits human epithelial Na+ channels by binding at a subunit interface of extracellular domains.
      ,
      • Collier D.M.
      • Snyder P.M.
      Identification of epithelial Na+ channel (ENaC) intersubunit Cl inhibitory residues suggests a trimeric αγβ channel architecture.
      ). The corresponding region in ASIC1 has been implicated in H+ sensing and desensitization (
      • Jasti J.
      • Furukawa H.
      • Gonzales E.B.
      • Gouaux E.
      Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH.
      ,
      • Li T.
      • Yang Y.
      • Canessa C.M.
      Asn415 in the β11-β12 linker decreases proton-dependent desensitization of ASIC1.
      ,
      • Li T.
      • Yang Y.
      • Canessa C.M.
      Leu85 in the β1-β2 linker of ASIC1 slows activation and decreases the apparent proton affinity by stabilizing a closed conformation.
      ,
      • Springauf A.
      • Bresenitz P.
      • Gründer S.
      The interaction between two extracellular linker regions controls sustained opening of acid-sensing ion channel 1.
      ). This solvent-accessible region appears to be an excellent target for the development of therapeutic agents that modulate activities of these related channels.
      The activating effect of Zn2+ on ENaC depends on the presence of Na+ self-inhibition (FIGURE 3, FIGURE 4, FIGURE 5) (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition.
      ). External Zn2+ may disrupt either Na+ binding to its receptor site or Na+-induced conformational changes. Our observations that mutations at the implicated Zn2+-binding sites (γH193A, γH200A, γH202A, and γH88A) did not alter the Na+ self-inhibition response suggest that the binding sites for Zn2+ and Na+ are distinct. The lack of effect on the apparent affinity for Zn2+ activation of a mutation of a critical site for Na+ self-inhibition (γH239A) also supports the notion of distinct Zn2+- and Na+-binding sites. Therefore, it is likely that Zn2+ binding interferes with Na+-induced motions within the ECD. Similar to Zn2+, several extracellular factors such as proteases, Cl, H+, and small molecules are thought to regulate ENaC gating in part by altering the Na+ self-inhibition response (
      • Collier D.M.
      • Snyder P.M.
      Extracellular chloride regulates the epithelial sodium channel.
      ,
      • Molina R.
      • Han D.Y.
      • Su X.F.
      • Zhao R.Z.
      • Zhao M.
      • Sharp G.M.
      • Chang Y.
      • Ji H.L.
      Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition.
      ,
      • Collier D.M.
      • Snyder P.M.
      Identification of epithelial Na+ channel (ENaC) intersubunit Cl inhibitory residues suggests a trimeric αγβ channel architecture.
      ,
      • Chraïbi A.
      • Horisberger J.D.
      Sodium self inhibition of human epithelial sodium channel: temperature dependence and effect of extracellular proteases.
      ,
      • Sheng S.
      • Carattino M.D.
      • Bruns J.B.
      • Hughey R.P.
      • Kleyman T.R.
      Furin cleavage activates the epithelial Na+ channel by relieving Na+ self-inhibition.
      ). It would be of great interest to determine whether other extracellular regulators affect Na+ self-inhibition in a similar manner to Zn2+.
      Recent studies suggest that ENaCs may belong to ligand-gated channels, like other members of the ENaC/degenerin family (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition.
      ,
      • Kashlan O.B.
      • Kleyman T.R.
      ENaC structure and function in the wake of a resolved structure of a family member.
      ,
      • Horisberger J.D.
      • Chraïbi A.
      Epithelial sodium channel: a ligand-gated channel?.
      ). Multiple external regulators of ENaC, including transition metals, Cl, H+, and other small molecules, exert their effects on ENaC gating by binding to regions within the ECD that are structurally distant from the channel pore (
      • Collier D.M.
      • Snyder P.M.
      Extracellular chloride regulates the epithelial sodium channel.
      ,
      • Chen J.
      • Myerburg M.M.
      • Passero C.J.
      • Winarski K.L.
      • Sheng S.
      External Cu2+ inhibits human epithelial Na+ channels by binding at a subunit interface of extracellular domains.
      ,
      • Molina R.
      • Han D.Y.
      • Su X.F.
      • Zhao R.Z.
      • Zhao M.
      • Sharp G.M.
      • Chang Y.
      • Ji H.L.
      Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition.
      ,
      • Collier D.M.
      • Snyder P.M.
      Identification of epithelial Na+ channel (ENaC) intersubunit Cl inhibitory residues suggests a trimeric αγβ channel architecture.
      ). These gating regulators are expected to remotely influence the channel gate through a series of conformational changes. The identification of these allosteric regulatory sites will advance our understanding regarding mechanisms by which the binding of allosteric regulators alters channel gating.
      In summary, this study provides novel insights into the structural basis for Zn2+ regulation of ENaC. Our results suggest that external Zn2+ regulates ENaC activity by binding to multiple extracellular sites, including a high-affinity activating site in the finger subdomain and a low-affinity inhibitory site in the palm subdomain of the γ-subunit. These findings advance our understanding of the regulation of ENaC gating by extracellular factors.

      Acknowledgments

      We thank Dr. Thomas R. Kleyman for helpful discussions and Brandon M. Blobner for oocyte preparation.

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