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

      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.

      EXPERIMENTAL PROCEDURES

       Site-directed Mutagenesis

      Point or multiple mutations were generated in mouse γENaC (γmENaC) cDNA in the pBluescript SK vector (Stratagene, La Jolla, CA) using the QuikChange II XL site-directed mutagenesis kit (Stratagene). Following mutagenesis, cDNAs were validated by sequencing. Wild-type ENaC α-, β-, and γ-subunit and mutant ENaC γ-subunit cRNAs were made using T3 RNA polymerase (Ambion), purified using an RNA purification kit (Qiagen), and quantified by spectrophotometry.

       ENaC Expression and Two-electrode Voltage Clamp

      ENaC expression in Xenopus oocytes and two-electrode voltage clamp were performed as reported previously (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      External nickel inhibits epithelial sodium channel by binding to histidine residues within the extracellular domains of α- and γ-subunits and reducing channel open probability.
      ). Stage V and VI oocytes free of follicle cell layers were injected with 1 ng of cRNA for each mENaC subunit per oocyte and incubated at 18 °C in modified Barth's saline (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 15 mm HEPES, 0.3 mm Ca(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4, 10 μg/ml sodium penicillin, 10 μg/ml streptomycin sulfate, and 100 μg/ml gentamycin sulfate, pH 7.4).
      All experiments were carried out at room temperature (20–24 °C). Two-electrode voltage clamp was performed using either an Axoclamp 900A amplifier (Molecular Devices, Sunnyvale, CA) or a TEV-200 voltage clamp amplifier (Dagan Corp., Minneapolis, MN) and a DigiData 1440A interface (Molecular Devices). Data acquisition and analyses were done with pCLAMP 8 or 9 (Molecular Devices). Oocytes were placed in a recording chamber from Warner Instruments (Hamden, CT) and perfused with bath solutions at a constant flow rate of 12–15 ml/min. Oocytes were continuously clamped at −60 or −100 mV as specified in the figure legends.

       Na+ Self-inhibition Response

      Na+ self-inhibition responses were examined as described previously (
      • Chraïbi A.
      • Horisberger J.D.
      Sodium self inhibition of human epithelial sodium channel: temperature dependence and effect of extracellular proteases.
      ,
      • Sheng S.
      • Bruns J.B.
      • Kleyman T.R.
      Extracellular histidine residues crucial for Na+ self-inhibition of epithelial Na+ channels.
      ). Briefly, Na+ self-inhibition was determined by measuring the decrease in current from the peak (Ipeak) to a steady state (Iss) elicited by a rapid increase in extracellular Na+ concentration from 1 to 110 mm. The [Na+] jump was done by rapidly replacing a 1 mm Na+ bath solution (NaCl-1 containing 1 mm NaCl, 109 mm N-methyl-d-glucamine, 2 mm KCl, 2 mm CaCl2, and 10 mm HEPES, pH 7.4) with a 110 mm Na+ bath solution (NaCl-110 containing 110 mm NaCl, 2 mm KCl, 2 mm CaCl2, and 10 mm HEPES, pH 7.4). Rapid solution exchange was performed with a 16-channel Teflon valve perfusion system (AutoMate Scientific, Inc., Berkeley, CA). The magnitude of Na+ self-inhibition was represented by the ratio of amiloride-sensitive Iss and Ipeak.

       Zn2+ Dose Responses

      A 1 m ZnCl2 stock solution (super pure, >99.999%, Sigma-Aldrich) was prepared in water and diluted to the desired concentrations in NaCl-110 bath solution. To examine Zn2+ dose responses, oocytes expressing mENaCs were continuously clamped at −60 mV while Zn2+ was applied at increasing concentrations in the range of 0.1 μm to 5 mm. Due to the limited solubility of ZnCl2 in NaCl-110 bath solution, Zn2+ concentrations >5 mm were not tested. At the end of an experiment, 10 μm amiloride was added to the bath to determine the amiloride-sensitive current. The changes in amiloride-sensitive currents in response to Zn2+ applications were used to analyze the dose-response relationship.
      Because the amiloride-sensitive current demonstrated a biphasic dose-response relationship with [Zn2+], we assumed that ENaCs possess a high-affinity activating site and a low-affinity inhibitory site for Zn2+. Thus, the normalized currents in the presence of increasing concentrations of Zn2+ were fitted with Equation 1,
      IR=(Imax-Imin)(C/(C+EC50))(IC50/(C+IC50))+Imin
      (Eq. 1)


      where IR is the relative current at certain [Zn2+] (i.e. current in the presence of Zn2+ normalized to current in the absence of Zn2+); Imax and Imin are the maximal and minimal normalized currents, respectively; C is the Zn2+ concentration; and EC50 and IC50 are the concentrations with 50% effects for the activating and inhibitory phases, respectively.

       Statistical Analysis

      Data are presented as means ± S.E. To minimize bias from batch-to-batch variation in oocytes, the significance in the differences between mutant and WT channels was always analyzed from data obtained in the same batches of oocytes. Student's t tests were used for comparison, and the significance levels are specified below and in the figure legends. Curve fittings were performed with OriginPro 8.5 (OriginLab, Northampton, MA).

      RESULTS

       External Zn2+ Regulates mENaC by Interacting at Distinct Sites

      Because extracellular Zn2+ activates ENaC by relieving Na+ self-inhibition (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition.
      ), the Na+ self-inhibition response and the effect of Zn2+ were examined sequentially in each oocyte. The Na+ self-inhibition response reflects a decrease in current from a peak (Ipeak) to a steady state (Iss), which is elicited by a rapid increase in the extracellular Na+ concentration from 1 to 110 mm (Fig. 1). As currents approached a steady-state level, increasing concentrations of Zn2+ were applied, and changes in current were measured. Increasing extracellular [Zn2+] in the range of ∼0.1–100 μm led to a dose-dependent increase in current. The current fell when extracellular [Zn2+] was increased above 100 μm. The maximal current in the presence of Zn2+ (Iznm) was essentially identical to the Ipeak observed following the increase in extracellular [Na+] (Iznm/Ipeak = 1.00 ± 0.01, n = 32), consistent with the notion that Zn2+ relieves ENaC from inhibition by external Na+.
      Figure thumbnail gr1
      FIGURE 1External Zn2+ activates αβγmENaC with a bell-shaped dose-response. A, a representative trace for Na+ self-inhibition response and the effect of Zn2+ on mENaC. The peak current (Ipeak), steady-state current (Iss), and the maximal current in the presence of Zn2+ (Iznm) are indicated by arrows. White, black, and gray bars indicate the time periods when oocytes were bathed in low Na+ (NaCl-1), high Na+ (NaCl-110), and 10 μm amiloride solutions, respectively. The concentrations of Zn2+ in NaCl-110 bath solution are shown by stepped lines and numbers above. This recording represents 39 independent observations in six batches of oocytes. B, dose response of WT mENaC to Zn2+. Relative currents are ratios of amiloride-sensitive currents obtained in the presence of Zn2+ and the currents measured immediately prior to Zn2+ application. The line is from a best fit of the data with the two-site model described under “Experimental Procedures” (R2 = 0.995). The parameters were as follows: Imax = 1.68; EC50 = 2.1 μm; IC50 = 2.1 mm; and Imin = 0.97. The averaged data are from six observations in one batch of oocytes, representative of six independent experiments performed in different batches of oocytes.
      Fig. 1B illustrates the Zn2+ dose-response relationship. Mean amiloride-sensitive currents in the presence of increasing extracellular [Zn2+] were normalized to the base-line current. Similar to our previous report (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition.
      ), we observed a bell-shaped dose response to Zn2+. This relationship fits a model of ENaC possessing a high-affinity Zn2+ activating site and a low-affinity Zn2+ inhibitory site (R2 = 0.997, Equation 1, see “Experimental Procedures”) with an estimated EC50 and IC50 of 2.1 ± 0.1 μm (n = 6) and 2.1 ± 0.1 mm (n = 6), respectively. The maximal relative current observed at 100 μm Zn2+ was 1.68 ± 0.07 (n = 6).

       Ala Substitutions of Selected His Residues in the Extracellular Region of γmENaC Alter the Effect of Zn2+

      There are a total of 10 His residues within the ECD of the γ-subunit. Four residues are located within the finger subdomain, three in the β-ball subdomain, two in the palm subdomain, and one in the thumb subdomain (Fig. 2). We individually mutated these His residues to Ala and expressed the mutant γ-subunits together with WT α- and β-subunits in oocytes. We sequentially examined Na+ self-inhibition and the response to Zn2+ (FIGURE 3, FIGURE 4) to determine whether a mutation selectively disrupted the effect of Zn2+ on channel activity or secondarily altered the Zn2+ effect by modifying the Na+ self-inhibition response.
      Figure thumbnail gr2
      FIGURE 2Mapping the extracellular His residues of γmENaC on the cASIC1 structure. A structural model for the ECD of one ASIC1 subunit was generated from ordinates in the Protein Data Bank (code 3HGC) using PyMOL 1.3 (
      • DeLano W.L.
      ). Secondary structures in the model are shown as gray ribbons and lines. Dashed lines encircle four of the five subdomains within the ECD, and the left-out region belongs to the palm subdomain. Extracellular ends of the two transmembrane domains (TM1 and TM2) are shown to reveal the covalent connections between the transmembrane domains and ECD. The black spheres show the locations of α-carbons of ASIC1 residues that are homologous to the His residues of γmENaC, based on sequence alignments. The locations of the four finger subdomain γHis residues are less certain than those of other residues, given the poor homology of this region among ENaC/degenerin family members. They serve as possible locations from one particular sequence alignment.
      Figure thumbnail gr4
      FIGURE 4Mutation-induced changes in the magnitude of Na+ self-inhibition and the maximal Zn2+ activation of mENaC. Iss/Ipeak values are inversely proportional to the magnitude of Na+ self-inhibition. Iznm values represent the maximal relative currents in the presence of Zn2+. They were typically observed with 100 μm Zn2+ except for the γH88A mutant, which had Iznm at 1 mm. Note that Iznm is the observed value and different from the Imax () that was derived from a best fit of the dose-response data. Black bars indicate that the values of mutants were significantly different (p < 0.01) from those of WT (n = 5–10), obtained in the same batch of oocytes. The dashed lines show the mean values pooled from all oocytes expressing WT mENaC obtained in the analyses of these mutants (Iss/Ipeak = 0.60 ± 0.01, Iznm = 1.73 ± 0.03, n = 32).
      Figure thumbnail gr3
      FIGURE 3Mutations of five γHis residues differentially alter the effect of Zn2+. Representative traces for current changes in responses to [Na+] increases and Zn2+ applications are from oocytes expressing αβγmENaC (WT), αβγH88A, αβγH193A, αβγH200A, αβγH202A, and αβγH239A mENaCs. A simplified protocol for examining the effects of Zn2+ at only six concentrations compared with the one in was used for both WT and mutant channels. The recording portions with 10 μm amiloride at the end of the experiments were omitted for clarity. These recordings represent at least six observations.
      Ala substitutions at 4 of the 10 extracellular γ-subunit His residues (γH193A, γH200A, γH202A, and γH239A) significantly reduced maximal ENaC activation by Zn2+ (FIGURE 3, FIGURE 4). Interestingly, all four of the extracellular His residues whose mutations reduced Zn2+ activation are within the predicted finger subdomain of the channel. In addition to suppressing Zn2+ activation, γH239A also diminished Na+ self-inhibition, similar to what was previously observed when γHis239 was substituted with other residues (
      • Sheng S.
      • Bruns J.B.
      • Kleyman T.R.
      Extracellular histidine residues crucial for Na+ self-inhibition of epithelial Na+ channels.
      ,
      • Winarski K.L.
      • Sheng N.
      • Chen J.
      • Kleyman T.R.
      • Sheng S.
      Extracellular allosteric regulatory subdomain within the γ-subunit of the epithelial Na+ channel.
      ). As the γH239A mutant was relieved of Na+ self-inhibition prior to the addition of Zn2+, it is not possible to determine whether this residue is involved in Zn2+ binding to the channel. On the other hand, γH193A, γH200A, and γH202A did not significantly alter the Na+ self-inhibition response, suggesting that these three His residues in the finger subdomain have specific roles in Zn2+ activation of ENaC.
      Although γH88A did not affect the maximal Zn2+ activation (p > 0.05 versus WT) or the Na+ self-inhibition response, it converted the Zn2+ dose response from bell-shaped to monophasic by selectively eliminating the inhibitory effect of high [Zn2+] (FIGURE 3, FIGURE 4). These results are consistent with a mutation-induced loss of a low-affinity Zn2+-binding site that mediates channel inhibition at high [Zn2+]. One mutation (γH283A) moderately increased the maximal Zn2+ activation (p < 0.01 versus WT) (Fig. 4). Collectively, these observations suggest that γHis88 within the palm subdomain and γHis193, γHis200, and γHis202 within the finger subdomain are involved in Zn2+-dependent regulation of ENaC.

       Mutation of γHis193, γHis200, or γHis202 within the Finger Subdomain Reduces the Apparent Zn2+ Binding Affinity of the Activating Site

      We analyzed the responses of WT and mutant channels to increasing [Zn2+] using a two-site model. Representative dose-response curves are shown in Fig. 5, and derived values for EC50 and IC50 are shown in Table 1. Our results show that γH88A did not affect the EC50 for the activating site, although it essentially eliminated the decrease in channel activity observed with higher [Zn2+]. In contrast, γH193A, γH200A, and γH202A reduced the sensitivity of mENaC to activation by extracellular Zn2+ and did not affect the inhibitory effect of high [Zn2+], leading to increased EC50 values and no change in the IC50 relative to WT. The results imply that γH193A, γH200A, and γH202A selectively reduce the apparent affinity of the potentiating site for Zn2+ while not interfering with the low-affinity Zn2+ inhibitory site.
      Figure thumbnail gr5
      FIGURE 5Ala substitutions of five γHis residues result in distinct changes in the Zn2+ dose response. Dose-response data are from the experiments performed as shown in (n = 34 for WT and 6–15 for mutants). Curve fittings were done in the same way as described in the legend to . Parameters from the best fits of the averaged data were as follows: Imax = 1.77, EC50 = 2.1 μm, and IC50 = 1.9 mm for WT; Imax = 1.79, EC50 = 2.4 μm, and IC50 > 1 m for γH88A; Imax = 1.55, EC50 = 4.6 μm, and IC50 = 1.9 mm for γH193A; Imax = 1.59, EC50 = 5.9 μm, and IC50 = 3.6 mm for γH200A; Imax = 1.50, EC50 = 6.1 μm, and IC50 = 3.0 mm for γH202A; and Imax = 1.11, EC50 = 1.0 μm, and IC50 = 1.6 mm for γH239A. Fitting parameters from individual oocytes expressing WT and mutants (means ± S.E.) were compared for statistical differences ().
      TABLE 1Fitting parameters for Zn2+ dose responses
      OocytesImaxEC50IC50
      μmmm
      WT441.76 ± 0.032.10 ± 0.122.13 ± 0.14
      γH88A101.79 ± 0.092.58 ± 0.32>1000
      γH193A61.54 ± 0.03
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      4.59 ± 0.46
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      1.97 ± 0.28
      γH200A141.59 ± 0.03
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      5.94 ± 0.27
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      3.69 ± 0.02
      γH202A151.50 ± 0.04
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      6.29 ± 0.56
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      3.22 ± 0.36
      γH239A91.18 ± 0.02
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      3.03 ± 0.771.09 ± 0.24
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      γH282A101.69 ± 0.091.79 ± 0.311.61 ± 0.21
      γH283A52.20 ± 0.09
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      2.11 ± 0.311.31 ± 0.10
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      γH338A51.88 ± 0.062.02 ± 0.373.10 ± 0.63
      γH364A61.74 ± 0.072.44 ± 0.251.68 ± 0.28
      γH434A121.61 ± 0.071.91 ± 0.241.71 ± 0.18
      γH200A/γH202A91.45 ± 0.03
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      6.74 ± 0.52
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      2.46 ± 0.36
      γH193A/γH200A/γH202A61.48 ± 0.03
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      6.41 ± 0.48
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      1.34 ± 0.23
      γH88A/γH200A/γH202A61.81 ± 0.1120.4 ± 3.00
      The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      >1000
      a The values were significantly different from the WT values in the same batch of oocytes (p < 0.01).
      We also examined whether substitutions at multiple His residues led to further increases in the EC50 for Zn2+. The Zn2+ dose-response relationships of γH200A/γH202A and γH193A/γH200A/γH202A were similar to those found in single mutants (Fig. 6). Thus, all three His residues within the finger subdomain are required for Zn2+-dependent channel activation. The lack of an additive effect of multiple His substitutions suggests that these His residues participate in a common Zn2+-binding site.
      Figure thumbnail gr6
      FIGURE 6Effects of Zn2+ on double and triple γHis mutations. Representative traces are from oocytes expressing αβγH200A/γH202A (A; n = 9), αβγH193A/γH200A/γH202A (B; n = 6), and αβγH88A/γH200A/γH202A (C; n = 6) mENaCs. Experiments were performed as described in the legend to . Curve fittings in D and E were carried out as described in the legend to . Derived parameters in D were as follows: Imax = 1.82, EC50 = 2.1 μm, and IC50 = 2.7 mm for WT; Imax = 1.45, EC50 = 6.5 μm, and IC50 = 2.3 mm for γH200A/γH202A; and Imax = 1.48, EC50 = 6.5 μm, and IC50 = 1.2 mm for γH193A/γH200A/γH202A. Parameters in E were as follows: Imax = 1.79, EC50 = 2.4 μm, and IC50 > 1 m for γH88A and Imax = 1.72, EC50 = 18.0 μm, and IC50 > 1 m for γH88A/γH200A/γH202A. Parameters from individual oocytes are shown in .
      In addition to increasing the EC50 values of Zn2+ activation, γH193A, γH200A, γH202A, γH200A/γH202A, and γH193A/γH200A/γH202A also reduced the magnitude of the increase in current in response to Zn2+ (Imax) (Table 1). The reduced Imax observed in the mutants could be explained either by a reduction in the efficacy of channel activation by Zn2+ or by the presence of an inhibitory effect that limits the extent of channel activation. To distinguish these two possibilities, we examined channels with His substitutions at Zn2+ activating sites (γH200A/γH202A) and the Zn2+ inhibitory site (γH88A). As shown in Fig. 6E, γH88A/γH200A/γH202A reduced Zn2+ sensitivity by 8-fold compared with γH88A channels (EC50 = 20.4 ± 3.0 μm (n = 6) versus 2.6 ± 0.3 μm (n = 10); p < 0.001). However, both mutants had similar Imax values (1.81 ± 0.11 (n = 6) versus 1.79 ± 0.09 (n = 10); p > 0.05), demonstrating that mutations of the Zn2+ activating sites within the finger subdomain do not alter the magnitude of the maximal Zn2+ activation when the low-affinity Zn2+ inhibitory site is not present. Considered together, these results are consistent with the idea that γH200A/γH202A reduces the apparent Zn2+ binding affinity for the activating site without altering its efficacy. The inhibitory site containing γHis88 limits the efficacy of channel activation by Zn2+.

       γHis239 Does Not participate in Zn2+ Binding

      The γH239A mutation eliminated both Na+ self-inhibition and Zn2+ activation of ENaC (FIGURE 3, FIGURE 4, FIGURE 5). As Zn2+ activates ENaC through relief of Na+ self-inhibition, it is unclear whether γHis239 is a Zn2+-binding site similar to the other finger subdomain His residues or whether the γH239A mutation simply prevents channel inhibition by external Na+. To distinguish between these two possibilities, additional mutations were added to restore Na+ self-inhibition in the setting of the γH239A mutant. Previously, we found that the G481A mutation within the α-subunit greatly enhanced the Na+ self-inhibition response (
      • Maarouf A.B.
      • Sheng N.
      • Chen J.
      • Winarski K.L.
      • Okumura S.
      • Carattino M.D.
      • Boyd C.R.
      • Kleyman T.R.
      • Sheng S.
      Novel determinants of epithelial sodium channel gating within extracellular thumb domains.
      ). Channels bearing both the αG481A and γH239A mutations exhibited a robust Na+ self-inhibition response and a biphasic response to Zn2+ (Fig. 7). Both αG481A and αG481A/γH239A channels had Imax values that were significantly greater than the WT values (3.4 ± 0.2 (n = 6) and 3.7 ± 0.2 (n = 6), respectively), presumably as a result of the enhanced Na+ self-inhibition. The EC50 for Zn2+ activation of αG481A/γH239A was similar to that of WT (4.2 ± 0.5 μm (n = 6) versus WT; p > 0.05), as was the IC50 of the Zn2+ inhibitory response (1.2 ± 0.0 mm (n = 6) versus WT; p > 0.05). Thus, γH239A did not weaken the apparent Zn2+ affinity of either the high- or low-affinity site.
      Figure thumbnail gr7
      FIGURE 7Na+ self-inhibition-enhancing mutation αG481A “restores” Zn2+ activation that is diminished by γH239A. A, representative recording in oocytes expressing αG481Aβγ mENaC (n = 6). B, representative recording of αG481A/βγH239A mENaC (n = 6). C, dose-response curves. Fitting parameters were as follows: Imax = 1.80, EC50 = 2.9 μm, and IC50 = 1.7 mm for WT; Imax = 3.4, EC50 = 9.6 μm, and IC50 = 1.8 mm for αG481Aβγ; and Imax = 3.7, EC50 = 4.2 μm, and IC50 = 1.2 mm for αG481A/βγH239A.

       Region Surrounding γHis88 Is a Determinant of the Low-affinity Inhibitory Site for Zn2+

      Next, the importance of the palm subdomain γHis88 and surrounding region was examined to explore the nature of the low-affinity Zn2+ inhibitory site. As shown in FIGURE 3, FIGURE 4, FIGURE 5, mutation of γHis88 to Ala prevented ENaC inhibition by high concentrations of Zn2+. Mutation of γHis88 to either Arg or Asp also rendered the Zn2+ dose responses monophasic and did not significantly alter the Na+ self-inhibition response (Fig. 8, A and B). These results suggest that a His residue at this position is an essential determinant of the Zn2+ inhibitory site.
      Figure thumbnail gr8
      FIGURE 8Mutations of γHis88 and γAsp516 eliminate the inhibitory component in the Zn2+ dose response. A, traces for γH88R and γH88D. B, dose-response curves of γHis88 mutants. Fitting parameters were as follows: Imax = 1.62, EC50 = 1.9 μm, and IC50 = 72.8 mm for αβγH88D and Imax = 1.52, EC50 = 1.9 μm, and IC50 = 96.3 mm for αβγH88R. C, a model of cASIC1 was generated from coordinates in the Protein Data Bank (code 3HGC) using PyMOL 1.3 (
      • DeLano W.L.
      ) to show the local structure within the ECD at the interface of subunits B (dark gray) and C (light gray). Black lines depict residues that are within 6 Å of Ala82, shown as a sphere. D, representative traces of γD516A and γD516R. E, dose-response curves of γD516A and γD516R. Fitting parameters were as follows: Imax = 1.51, EC50 = 1.6 μm, and IC50 = 16.6 mm for αβγD516A and Imax = 1.44, EC50 = 3.8 μm, and IC50 = 13.9 mm for αβγD516R. F, trace of αβγH88A/γD516A. G, dose responses for WT and αβγH88A/γD516A mENaCs. Fitting parameters were as follows: Imax = 1.83, EC50 = 2.4 μm, and IC50 = 2.4 mm for WT and Imax = 1.74, EC50 = 2.8 μm, and IC50 > 1 m for αβγH88A/γD516A.
      Zn2+ is often coordinated by multiple neighboring amino acid residues in metal-binding proteins (
      • 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.
      ). To define additional residues that contribute to the low-affinity Zn2+ inhibitory site, we searched for additional potential Zn2+-binding residues using sequence alignments of ENaC and ASIC and the resolved cASIC1 structure (
      • Gonzales E.B.
      • Kawate T.
      • Gouaux E.
      Pore architecture and ion sites in acid-sensing ion channels and P2X receptors.
      ). γHis88 aligns with Ala82 in cASIC1, which is located immediately after the first β-strand of ECD (β1) within the palm subdomain (Fig. 8C). Multiple regions within the same subunit (strand β1 and loops β1-β2, β11-β12, and β5-β6) and an adjacent subunit (N-terminal end of helix α4 and loop α5-β10) are in close proximity to Ala82 in cASIC1. Eleven residues in these regions are within 6 Å of the Ala82 (Fig. 8C). Among these residues, only Ala413 and Met364 have homologous residues in mENaC that are often found within a Zn2+ coordination shell (γAsp516 and βGlu444, respectively). We examined whether these sites are involved in ENaC regulation by Zn2+. As shown in Fig. 8 (D and E), γD516A and γD516R largely prevented ENaC inhibition by high [Zn2+], similar to what we observed with the γH88A mutant (Fig. 5). Mutation of the other residue that was predicted to be in close proximity to γHis88 (βE444A) did not significantly alter the Zn2+ effects on ENaC (data not shown). The proximity of γAsp516 to γHis88 and their similar mutation-induced changes in the Zn2+ response suggest that the two residues share a functional role in constituting the low-affinity Zn2+ inhibitory site.
      The residues corresponding to γHis88 and γAsp516 in cASIC1 (Ala82 and Ala413) are located at a subunit interface (Fig. 8C). According to the suggested counterclockwise arrangement (viewed from above) of the ENaC α-, β-, and γ-subunits (
      • 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.
      ), γHis88 (Ala82 in cASIC1) and γAsp516 (Ala413 in cASIC1) are expected to interact with residues within the thumb subdomain of βENaC. There are five negatively charged βENaC residues within loops β9-α4 and α5-β10 of the thumb subdomain: βGlu354, βAsp365, βGlu436, βGlu444, and βGlu448. Given their locations within flexible loops, they might be in close proximity to γHis88 and γAsp516 and contribute to the low-affinity inhibitory site. Mutations of these β-subunit residues to Ala did not alter the channel's response to Zn2+ (data not shown). Thus, it is unlikely that these residues within the thumb domain of βENaC are involved in Zn2+ regulation of the channel.

      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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