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External Cu2+ Inhibits Human Epithelial Na+ Channels by Binding at a Subunit Interface of Extracellular Domains*

  • Jingxin Chen
    Affiliations
    Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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  • Mike M. Myerburg
    Affiliations
    Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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  • Christopher J. Passero
    Affiliations
    Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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  • Katie L. Winarski
    Affiliations
    Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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  • Shaohu Sheng
    Correspondence
    To whom correspondence should be addressed: Renal-Electrolyte Division, University of Pittsburgh, S929 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9295; Fax: 412-383-8956.
    Affiliations
    Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01 ES014701, K08 HL087932, P30 DK072506, and P30 DK079307. This work was also supported by a research grant from Dialysis Clinic, Inc. and the Cystic Fibrosis Foundation RDP to the University of Pittsburgh.
Open AccessPublished:June 09, 2011DOI:https://doi.org/10.1074/jbc.M111.232058
      Epithelial Na+ channels (ENaCs) play an essential role in the regulation of body fluid homeostasis. Certain transition metals activate or inhibit the activity of ENaCs. In this study, we examined the effect of extracellular Cu2+ on human ENaC expressed in Xenopus oocytes and investigated the structural basis for its effects. External Cu2+ inhibited human αβγ ENaC with an estimated IC50 of 0.3 μm. The slow time course and a lack of change in the current-voltage relationship were consistent with an allosteric (non pore-plugging) inhibition of human ENaC by Cu2+. Experiments with mixed human and mouse ENaC subunits suggested that both the α and β subunits were primarily responsible for the inhibitory effect of Cu2+ on human ENaC. Lowering bath solution pH diminished the inhibition by Cu2+. Mutations of two α, two β, and two γ His residues within extracellular domains significantly reduced the inhibition of human ENaC by Cu2+. We identified a pair of residues as potential Cu2+-binding sites at the subunit interface between thumb subdomain of αhENaC and palm subdomain of βhENaC, suggesting a counterclockwise arrangement of α, β, and γ ENaC subunits in a trimeric channel complex when viewed from above. We conclude that extracellular Cu2+ is a potent inhibitor of human ENaC and binds to multiple sites within the extracellular domains including a subunit interface.

      Introduction

      The epithelial Na+ channel (ENaC)
      The abbreviations used are: ENaC
      epithelial Na+ channel
      hENaC
      human ENaC
      mENaC
      mouse ENaC
      ASIC
      acid sensing ion channel
      ECD
      extracellular domain
      MES
      2-(N-morpholino) ethanesulfonic acid
      MOPS
      3-(N-morpholino) propanesulfonic acid
      TTM
      tetrathiomolybdate.
      mediates Na+ transport across apical membranes of high resistance epithelia in kidney, colon, and lung. ENaC has important roles in the maintenance of extracellular fluid volume and the regulation of airway surface liquid volume (
      • Sheng S.
      • Johnson J.P.
      • Kleyman T.R.
      ). Alterations in ENaC activity have been associated with several human diseases. For example, enhanced ENaC activity is responsible for the hypertension seen in Liddle's syndrome, contributes to the mucociliary dysfunction seen in cystic fibrosis, and is believed to contribute to hypervolemia associated with nephrotic syndrome (
      • Bhalla V.
      • Hallows K.R.
      ,
      • Passero C.J.
      • Hughey R.P.
      • Kleyman T.R.
      ).
      A variety of intracellular and extracellular factors regulate ENaC activity by distinct mechanisms (
      • Garty H.
      • Palmer L.G.
      ). External amiloride analogs, cations, anions, nucleotides, serine proteases, and laminar shear stress inhibit or stimulate endogenous or exogenous ENaCs (
      • Van Driessche W.
      • Zeiske W.
      ,
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      ,
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      ,
      • Collier D.M.
      • Snyder P.M.
      ,
      • Collier D.M.
      • Snyder P.M.
      ,
      • Nie H.G.
      • Zhang W.
      • Han D.Y.
      • Li Q.N.
      • Li J.
      • Zhao R.Z.
      • Su X.F.
      • Peng J.B.
      • Ji H.L.
      ,
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ,
      • Carattino M.D.
      • Sheng S.
      • Kleyman T.R.
      ). All of these extracellular regulators appear to directly alter the activity of ENaCs in plasma membranes rather than affect channel subunit trafficking (
      • Sheng S.
      • Johnson J.P.
      • Kleyman T.R.
      ). Their primary targets likely reside within the characteristically large extracellular domains (ECDs) of ENaC subunits. This notion is in line with the well defined subdomains within the ECDs of the chicken acid-sensing ion channel 1 (cASIC1), a member of the ENaC/degenerin family, revealed in a crystal structure and the identification of proton binding sites within the ECDS (
      • Jasti J.
      • Furukawa H.
      • Gonzales E.B.
      • Gouaux E.
      ).
      We have previously examined the effects of the transition metals, Ni2+ and Zn2+, on ENaC activity. External Ni2+ inhibits and Zn2+ activates mouse ENaCs in Xenopus oocytes by directly interacting with the channels and altering channel gating (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      ,
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      ). Some of these metal effects are thought to be related to Na+ self-inhibition, a down-regulation of open probability (Po) by extracellular Na+ (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      ,
      • Yu L.
      • Eaton D.C.
      • Helms M.N.
      ). Yu et al. (
      • Yu L.
      • Eaton D.C.
      • Helms M.N.
      ) have also examined the effects of several transition metals on the single channel activity of native Xenopus ENaCs in A6 cells. These metals differentially affect Xenopus ENaC Po and channel number in membrane patches without changing the single channel conductance. However, the exact binding sites and detailed mechanisms for the metal effects on ENaCs remain largely unknown.
      Copper is the third most abundant trace metal in humans and has a variety of important biological functions. Excessive Cu2+ is highly toxic to cells, and its content in cells is carefully maintained at low levels. Indeed, Cu2+ is implicated in several human diseases such as Wilson disease, Menkes disease, neurodegenerative disorders, and cancers (
      • Ellingsen D.G.
      • Horn N.
      • Aaseth J.
      ,
      • Tisato F.
      • Marzano C.
      • Porchia M.
      • Pellei M.
      • Santini C.
      ). The therapeutic potential of copper chelators and copper complexes is being intensively investigated (
      • Tisato F.
      • Marzano C.
      • Porchia M.
      • Pellei M.
      • Santini C.
      ). In addition, particulate matters contain high amounts of transitional metals including copper. Soluble metals in airborne particles contribute to pulmonary and cardiovascular toxicity (
      • Adamson I.Y.
      • Prieditis H.
      • Vincent R.
      ,
      • Prieditis H.
      • Adamson I.Y.
      ). Recent studies suggest that copper nanoparticles are highly toxic (
      • Karlsson H.L.
      • Cronholm P.
      • Gustafsson J.
      • Möller L.
      ). The underlying mechanisms for the harmful effects of Cu2+ are not fully understood. Many studies have suggested that certain metals exert their toxic effects in part by altering functions of ion channels or transporters (
      • Kiss T.
      • Osipenko O.N.
      ,
      • Restrepo-Angulo I.
      • De Vizcaya-Ruiz A.
      • Camacho J.
      ). Clearly, a better understanding of the interactions between copper and biological molecules is crucial to an elucidation of its physiological, pathological, and toxicological roles in human health.
      In this report, we examined the effects of external Cu2+ on amiloride-sensitive Na+ currents in oocytes expressing αβγ human ENaC (hENaC) and probed the structural basis by site-directed mutagenesis. We found that external Cu2+ is a potent inhibitor of hENaC. The inhibitory effect of Cu2+ on hENaC depends on the α and β subunits. The most important site for Cu2+ inhibition was identified at the α/β subunit interface.

      DISCUSSION

      In this study, we found that external Cu2+ inhibited human αβγ ENaC in both Xenopus oocytes and human airway epithelia. External Cu2+ at 10 μm does not inhibit mouse (Fig. 4) or rat ENaC.
      J. Chen and S. Sheng, unpublished observations.
      Native Xenopus ENaCs in A6 cells are activated by extracellular Cu2+ (
      • Yu L.
      • Eaton D.C.
      • Helms M.N.
      ). Therefore, in the context of previous reports and our current observations, external Cu2+ appears to be a specific inhibitor of human ENaC among cloned ENaCs. The inhibitory effect of Cu2+ on the hENaC current in the human airway epithelia appeared to be smaller and weaker than that in oocytes (Fig. 11). We do not know the exact cause for the different responses to Cu2+. They could be related to certain experimental conditions utilized in the two systems, such as temperatures (i.e. 20–24 °C in oocytes and 37 °C in epithelia) and oxygen tensions, both of which regulate ENaC activity (
      • Chraïbi A.
      • Horisberger J.D.
      ,
      • Askwith C.C.
      • Benson C.J.
      • Welsh M.J.
      • Snyder P.M.
      ,
      • Davis I.C.
      • Matalon S.
      ). The differences in the response to Cu2+ could also reflect the inherent differences between native and heterologously expressed channels. It has been reported that ENaCs in oocytes and epithelial monolayers display different sensitivities to the peptide inhibitors derived from the inhibitory domains of α and γ mouse ENaCs (
      • Carattino M.D.
      • Sheng S.
      • Bruns J.B.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      ,
      • Bruns J.B.
      • Carattino M.D.
      • Sheng S.
      • Maarouf A.B.
      • Weisz O.A.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      ).
      The estimated IC50 of 0.31 μm for Cu2+ inhibition suggests that external Cu2+ is a potent hENaC inhibitor. A high affinity ENaC inhibitor may be useful in treating diseases associated with elevated ENaC-mediated Na+ absorption such as Liddle syndrome and cystic fibrosis (
      • Rossier B.C.
      • Pradervand S.
      • Schild L.
      • Hummler E.
      ,
      • Donaldson S.H.
      • Boucher R.C.
      ).
      Recent studies have established a critical role for ENaCs in the regulation of airway surface liquid volume and excessive activity of ENaCs in airways contributes to the pathogenesis of cystic fibrosis (
      • Donaldson S.H.
      • Boucher R.C.
      ). Impaired Na+ transport in alveoli leads to pulmonary edema (
      • Davis I.C.
      • Matalon S.
      ). Airborne particles contain a considerate amount of transition metals including copper. Upon contact with biological fluids, free metal ions can be released from the particles and cause local and even remote toxic effects (
      • Adamson I.Y.
      • Prieditis H.
      • Vincent R.
      ,
      • Prieditis H.
      • Adamson I.Y.
      ). We speculate that the inhibitory effect of Cu2+ on human ENaCs in lung epithelia may contribute to the toxicological symptoms caused by inhaled particulate matters. Cu2+, released from particulate matters, may worsen particle-induced pulmonary edema by inhibition of Na+ absorption in airways and alveoli.
      The main goal of this study was to probe the structural basis for hENaC inhibition by external Cu2+. Experiments with mixed human and mouse ENaC subunits demonstrate that α and β hENaC subunits are necessary for the specific response of hENaCs to Cu2+ (Fig. 4). Initial mutational screening of the hENaC-specific residues within α and β ECDs identified αHis468 and βHis159 as residues involved in Cu2+ inhibition (Fig. 5). The double mutant (αH468S-βH159D) converted the response to Cu2+ of the human channel to that of the mouse channel (FIGURE 4, FIGURE 5), suggesting that these two hENaC-specific residues are primarily responsible for the distinct response to 10 μm Cu2+ of human versus mouse ENaC. Subsequently, systematic screening of His residues within ECDs of α, β, and γ subunits identified additional four sites (αHis255, βHis160, γHis88, and γHis277) where mutations significantly reduced the inhibitory effect of 10 μm Cu2+ (Fig. 7). However, further analyses showed that αHis255 and γHis277 mutations significantly increased the magnitudes of Na+ self-inhibition and Cu2+-induced transient activation preceding the inhibitory effect (Figs. 7D and 10), suggesting indirect roles for both His residues in the Cu2+ inhibition. Another residue, γHis88, does not appear to have an essential role in Cu2+ inhibition, given the small effect of its mutation on Cu2+ inhibition (Fig. 7). On the contrary, αHis468, βHis159, and βHis160 mutations specifically reduced Cu2+ inhibition, without affecting the Na+ self-inhibition response and the transient activation by Cu2+. We conclude that these three His residues are involved in Cu2+ inhibition.
      Our data suggest that αHis468 has a key role in mediating Cu2+ inhibition. Taking advantage of the structural information for cASIC1 (
      • Jasti J.
      • Furukawa H.
      • Gonzales E.B.
      • Gouaux E.
      ,
      • Gonzales E.B.
      • Kawate T.
      • Gouaux E.
      ), we predicted that αHis468 and βGlu254 could contribute to a Cu2+-binding site at the α/β subunit interface. Mutational analyses confirmed the prediction (Fig. 9). The identification of αHis468/βGlu254 pair suggests a counterclockwise configuration of α, β, and γ subunits when viewed from above the channel (Fig. 9F). This subunit arrangement is in agreement with a recent report by Collier and Snyder (
      • Collier D.M.
      • Snyder P.M.
      ). However, we cannot rule out the presence of both the counterclockwise and clockwise subunit arrangements. Another limitation of the notion is that it is based on the assumption that ENaC, like ASIC1, has a trimeric architecture, which remains to be established experimentally.
      ENaC Po is regulated by a variety of extracellular factors that may share common pathways in their regulation of ENaC gating. Indeed, the effects of external Zn2+, H+, and Cl on ENaC activity rely on the existence of Na+ self-inhibition (
      • Sheng S.
      • Perry C.J.
      • Kleyman T.R.
      ,
      • Collier D.M.
      • Snyder P.M.
      ,
      • Collier D.M.
      • Snyder P.M.
      ). However, Cu2+ inhibition of hENaC does not depend on the existence of Na+ self-inhibition. In fact, the magnitude of Cu2+ inhibition was increased by Na+ self-inhibition eliminating mutation (γH233A) and reduced by Na+ self-inhibition enhancing mutation (αH255A). Therefore, Cu2+ likely inhibits the hENaC via a pathway distinct from that of Na+ self-inhibition. In contrast, the transient activation of hENaC by Cu2+ appears to result from a relief of Na+ self-inhibition, because Na+ self-inhibition eliminating (γH233A) or enhancing (αH255A) mutations diminished or enhanced its magnitude accordingly.
      In summary, we found that external Cu2+ is a high affinity inhibitor of human ENaC and identified a Cu2+-binding site at subunit interface within the extracellular domains. Structure-assisted mutational analyses suggest that a thumb domain His residue of α subunit and a palm domain Glu residue of the β subunit interact with a Cu2+ ion. This pairing (αHis468/βGlu254) requires a counterclockwise arrangement of α, β, and γ ENaC subunits when viewed from above.

      Acknowledgments

      We thank Dr. Thomas R. Kleyman for critical reading and comments on this manuscript, Dr. Joseph Pilewski at the Airway Epithelial Cell Culture Core at the University of Pittsburgh for providing human bronchial epithelial cultures, and Brandon M. Blobner for oocyte preparation.

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