Advertisement

Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains*

  • Ossama B. Kashlan
    Affiliations
    From the Departments of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
    Search for articles by this author
  • Cary R. Boyd
    Affiliations
    From the Departments of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
    Search for articles by this author
  • Christos Argyropoulos
    Affiliations
    From the Departments of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
    Search for articles by this author
  • Sora Okumura
    Affiliations
    From the Departments of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
    Search for articles by this author
  • Rebecca P. Hughey
    Affiliations
    From the Departments of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

    Cell Biology and Physiology, and University of Pittsburgh, Pittsburgh, Pennsylvania 15261
    Search for articles by this author
  • Michael Grabe
    Affiliations
    Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

    Computational and Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
    Search for articles by this author
  • Thomas R. Kleyman
    Correspondence
    To whom correspondence should be addressed: Renal-Electrolyte Division, A919 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261
    Affiliations
    From the Departments of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

    Cell Biology and Physiology, and University of Pittsburgh, Pittsburgh, Pennsylvania 15261
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants DK078734 (to O. B. K.), DK051391 (to T. R. K.), DK065161 (to T. R. K. and R. P. H.), and DK079307 (Pittsburgh Center for Kidney Research). This work was also supported by a scientist development grant from the American Heart Association and a grant from the PNC Charitable Trust Foundation (to M. G.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.
Open AccessPublished:September 03, 2010DOI:https://doi.org/10.1074/jbc.M110.167064
      The epithelial Na+ channel (ENaC) mediates the rate-limiting step in transepithelial Na+ transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.

      Introduction

      The epithelial Na+ channel (ENaC)
      The abbreviations used are: ENaC
      epithelial Na+ channel
      P8
      Ac-LPHPLQRL-amide
      ASIC1
      acid-sensing ion channel 1
      NLMR
      nonlinear mixed regression.
      is expressed at the apical surface of Na+-transporting epithelia such as the distal nephron of the kidney, distal colon, and lung alveoli and airway. In conjunction with the Na+/K+-ATPase, ENaC transfers Na+ from the luminal to the interstitial space. This transfer is crucial in regulating blood pressure through its role in renal Na+ absorption and in regulating airway surface liquid volume and mucociliary clearance through its role in airway Na+ absorption. In accord with its role in these processes, improper ENaC function is implicated in several disorders. There is a growing body of evidence that enhanced ENaC activity in the airways of individuals with cystic fibrosis contributes to depletion of airway surface liquids resulting in poor mucociliary clearance (
      • Rauh R.
      • Diakov A.
      • Tzschoppe A.
      • Korbmacher J.
      • Azad A.K.
      • Cuppens H.
      • Cassiman J.J.
      • Dötsch J.
      • Sticht H.
      • Korbmacher C.
      ,
      • Fajac I.
      • Viel M.
      • Gaitch N.
      • Hubert D.
      • Bienvenu T.
      ,
      • Sheng S.
      • Johnson J.P.
      • Kleyman T.R.
      ). In the kidney, increased levels of aldosterone activate ENaC and increase the reabsorption of filtered Na+ (
      • Bhalla V.
      • Hallows K.R.
      ). In both instances, increases in channel activity reflect, in part, enhanced channel proteolysis. Proteinuric states, characterized by excessive protein in the urine, are often accompanied by renal Na+ retention, volume expansion, and hypertension. Recent work indicates that volume expansion in proteinuric states reflects proteolytic activation of ENaC (
      • Kastner C.
      • Pohl M.
      • Sendeski M.
      • Stange G.
      • Wagner C.A.
      • Jensen B.
      • Patzak A.
      • Bachmann S.
      • Theilig F.
      ,
      • Passero C.J.
      • Hughey R.P.
      • Kleyman T.R.
      ,
      • Passero C.J.
      • Mueller G.M.
      • Rondon-Berrios H.
      • Tofovic S.P.
      • Hughey R.P.
      • Kleyman T.R.
      ).
      ENaC is a trimer composed of three homologous subunits, α, β, and γ (
      • Jasti J.
      • Furukawa H.
      • Gonzales E.B.
      • Gouaux E.
      ,
      • Staruschenko A.
      • Adams E.
      • Booth R.E.
      • Stockand J.D.
      ). ENaC subunits are members of the much larger ENaC/Degenerin family of ion channel proteins. These channels share a few salient features as follows: 1) most are gated by ligands and/or mechanical forces; 2) they are Na+-permeable and blocked by amiloride, a potassium-sparing diuretic; and 3) each subunit has two transmembrane helices (six transmembrane helices for the full channel), short intracellular N and C termini, and a large extracellular region comprised of several domains. Acid-sensing ion channels (ASIC) are also members of the ENaC/Degenerin family. The recently resolved structure of ASIC1 has provided important clues regarding the structural organization of ENaCs. Of note is that its extracellular region has well defined domains, termed finger, thumb, palm, knuckle, and β-ball.
      ENaC α and γ subunits undergo a very unusual form of regulatory processing. Each subunit can be cleaved at two (or more) distinct extracellular sites resulting in the liberation of a small stretch of amino acids and an increase in channel activity (
      • Bruns J.B.
      • Carattino M.D.
      • Sheng S.
      • Maarouf A.B.
      • Weisz O.A.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      ,
      • Hughey R.P.
      • Bruns J.B.
      • Kinlough C.L.
      • Harkleroad K.L.
      • Tong Q.
      • Carattino M.D.
      • Johnson J.P.
      • Stockand J.D.
      • Kleyman T.R.
      ). For both subunits, we have shown that synthetic peptides corresponding to these released tracts, and subsets thereof, are inhibitory (
      • Passero C.J.
      • Carattino M.D.
      • Kashlan O.B.
      • Myerburg M.M.
      • Hughey R.P.
      • Kleyman T.R.
      ,
      • Carattino M.D.
      • Passero C.J.
      • Steren C.A.
      • Maarouf A.B.
      • Pilewski J.M.
      • Myerburg M.M.
      • Hughey R.P.
      • Kleyman T.R.
      ,
      • Carattino M.D.
      • Sheng S.
      • Bruns J.B.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      ). We reasoned that the inhibitory peptides and proteolytically liberated fragments have similar binding sites and inhibitory mechanisms.
      In an effort to elucidate the mechanism of proteolytic activation of ENaC, we functionally characterized the binding site for an α subunit-derived 8-residue inhibitory peptide. To map sites within α ENaC that interact with this peptide, we systematically mutated individual residues within several peripheral regions of the α subunit to Trp and measured the effect of these mutations on peptide-dependent channel inhibition. As some mutations may indirectly affect the ability of the peptide to inhibit the channel, we analyzed our data within a thermodynamic framework that allowed us to deduce the direct effects of mutations on peptide-dependent channel inhibition. Using these data, we performed double mutant cycle experiments to identify pairwise interactions. We found two residues that interact with a site toward the N terminus of the peptide and one residue that interacts with the C terminus of the peptide. Our results suggest that the peptide binds to both the finger and thumb domains, with the N terminus of the peptide binding at the finger-thumb interface. Because the peptide is an allosteric inhibitor of ENaC, these data provide support for the importance of the finger-thumb interface in the mechanism of ENaC gating.

      DISCUSSION

      Proteolytic cleavage leading to channel activation is an unusual mechanism for channel regulation in biology. Although proteolytic cleavage has an important role in regulating ENaC, proteolysis may also regulate other members of the ENaC/Degenerin family (
      • Clark E.B.
      • Jovov B.
      • Rooj A.K.
      • Fuller C.M.
      • Benos D.J.
      ). For ENaC, both the α and γ subunits undergo proteolysis in association with channel activation (
      • Bruns J.B.
      • Carattino M.D.
      • Sheng S.
      • Maarouf A.B.
      • Weisz O.A.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      ,
      • Hughey R.P.
      • Bruns J.B.
      • Kinlough C.L.
      • Harkleroad K.L.
      • Tong Q.
      • Carattino M.D.
      • Johnson J.P.
      • Stockand J.D.
      • Kleyman T.R.
      ,
      • Carattino M.D.
      • Sheng S.
      • Bruns J.B.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      ,
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ). The activation of ENaC by proteolysis requires that a subunit be cleaved twice, releasing an intrinsic inhibitory tract. Peptides corresponding to the sequences of these excised tracts within the α and γ subunits inhibit ENaC currents (
      • Bruns J.B.
      • Carattino M.D.
      • Sheng S.
      • Maarouf A.B.
      • Weisz O.A.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      ,
      • Passero C.J.
      • Carattino M.D.
      • Kashlan O.B.
      • Myerburg M.M.
      • Hughey R.P.
      • Kleyman T.R.
      ,
      • Carattino M.D.
      • Passero C.J.
      • Steren C.A.
      • Maarouf A.B.
      • Pilewski J.M.
      • Myerburg M.M.
      • Hughey R.P.
      • Kleyman T.R.
      ,
      • Carattino M.D.
      • Sheng S.
      • Bruns J.B.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      ). These inhibitory tracts reside within the variable finger domains of these subunits and have no analogs within ASIC1 or other ENaC/Degenerin family members (
      • Jasti J.
      • Furukawa H.
      • Gonzales E.B.
      • Gouaux E.
      ).
      In this study, we identified two distinct regions that are likely involved in the binding of an 8-residue peptide derived from the α subunit inhibitory tract. We note that mutations at these sites may have indirectly affected peptide binding through distortions of structure or access to the binding site, and we interpret our results mindful of this limitation. These regions encompass residues 470–473 in the thumb domain, and residues 239–289 in the finger domain. Neither region is contiguous with the α subunit inhibitory tract (residues 206–231) or residues corresponding to P8 (211–218). We also identified specific pairwise interactions between ENaC and P8. As Arg-289 and Asp-473 both interact with the third position of the peptide, our data suggest that Arg-289 and Asp-473 are in close proximity and that the N-terminal region of the peptide binds at a thumb-finger interface. As Arg-289 follows a stretch of residues that our Na+ self-inhibition data suggest is helical, our data suggest that a putative loop containing Arg-289 is in close proximity to the top of the thumb. This suggests that ENaC shares some common structural features with ASIC1 within the finger domain despite a lack of sequence identity in this region, as the α2-α3 loop within the finger domain of ASIC is also in close proximity to its thumb domain. We also found that Gln-254 interacts with the eighth position of P8. If the peptide assumes an extended conformation in the bound state, our data suggest that Gln-254 is not adjacent to the thumb-finger interface.
      There is now strong evidence that P8 is an allosteric inhibitor of ENaC. First, we previously found that P8 binding is not voltage-dependent despite the fact that it is positively charged at pH 7.4, suggesting that P8 does not bind within the ion permeation pathway of the channel (
      • Carattino M.D.
      • Sheng S.
      • Bruns J.B.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      ). Second, we have shown that apparent P8 affinity is influenced by channel open probability (Fig. 1, A and B). Third, we have identified mutations at residues in the finger and the thumb domains of α ENaC that attenuate P8 affinity, suggesting that P8 binds in the periphery of the channel and far from the pore. The finding that P8 is an allosteric inhibitor of ENaC leads to the conclusion that P8 inhibits the channel by preferentially stabilizing the closed state of the channel. Because P8 is derived from the furin-excised inhibitory tract, we suggest that the inhibitory tract functions largely through similar mechanisms. Based on this reasoning, we propose that furin activates ENaC through α subunit cleavage by removing an intrinsic allosteric inhibitor from a site partially defined by the finger-thumb interface.
      In the course of this study, we measured the Na+ self-inhibition of α subunit Trp mutants within the finger and thumb domains. Interpretation of these results is complicated by the fact that we cannot readily distinguish between mutations that directly affect Na+ self-inhibition by altering Na+ binding and mutations that indirectly affect Na+ self-inhibition by altering the downstream allosteric machinery of the channel. For example, the βS518K pore mutant largely eliminates ENaC Na+ self-inhibition (
      • Winarski K.L.
      • Sheng N.
      • Chen J.
      • Kleyman T.R.
      • Sheng S.
      ). If this was evidence for a Na+-binding site nearby, Na+ self-inhibition would be predicted to be voltage-dependent, contrary to published work (
      • Bize V.
      • Horisberger J.D.
      ). The simplest explanation is that βS518K stabilizes the open state of the channel relative to the closed state, raising the thermodynamic barrier to channel closure by Na+. Mindful of this limitation, our Na+ self-inhibition data provide evidence for an α-helix that encompasses residues 271–285.
      The finger domains of members of the ENaC/Degenerin family of ion channels are hypervariable, which leads to the hypothesis that the finger domains of these proteins are functional modules. In the case of α ENaC, the finger domain appends protease sensitivity and possibly Na+ sensitivity as well. Given the modular finger hypothesis and the large number of sites in the finger domain where mutations altered Na+ self-inhibition, some of these sites may be directly involved in Na+ binding. Also, consideration of the P8-binding site within the finger and at the finger-thumb interface suggests that P8, and by extension the α-inhibitory tract, recruits much of the same allosteric machinery as used by Na+.

      REFERENCES

        • Rauh R.
        • Diakov A.
        • Tzschoppe A.
        • Korbmacher J.
        • Azad A.K.
        • Cuppens H.
        • Cassiman J.J.
        • Dötsch J.
        • Sticht H.
        • Korbmacher C.
        J. Physiol. 2010; 588: 1211-1225
        • Fajac I.
        • Viel M.
        • Gaitch N.
        • Hubert D.
        • Bienvenu T.
        Eur. Respir. J. 2009; 34: 772-773
        • Sheng S.
        • Johnson J.P.
        • Kleyman T.R.
        Alpern R.J. Hebert S.C. The Kidney, Physiology and Pathophysiology. 4th Ed. Elsevier Publishing, Philadelphia2008: 743-768
        • Bhalla V.
        • Hallows K.R.
        J. Am. Soc. Nephrol. 2008; 19: 1845-1854
        • Kastner C.
        • Pohl M.
        • Sendeski M.
        • Stange G.
        • Wagner C.A.
        • Jensen B.
        • Patzak A.
        • Bachmann S.
        • Theilig F.
        Am. J. Physiol. Renal Physiol. 2009; 296: F902-F911
        • Passero C.J.
        • Hughey R.P.
        • Kleyman T.R.
        Curr. Opin. Nephrol. Hypertens. 2010; 19: 13-19
        • Passero C.J.
        • Mueller G.M.
        • Rondon-Berrios H.
        • Tofovic S.P.
        • Hughey R.P.
        • Kleyman T.R.
        J. Biol. Chem. 2008; 283: 36586-36591
        • Jasti J.
        • Furukawa H.
        • Gonzales E.B.
        • Gouaux E.
        Nature. 2007; 449: 316-323
        • Staruschenko A.
        • Adams E.
        • Booth R.E.
        • Stockand J.D.
        Biophys. J. 2005; 88: 3966-3975
        • Bruns J.B.
        • Carattino M.D.
        • Sheng S.
        • Maarouf A.B.
        • Weisz O.A.
        • Pilewski J.M.
        • Hughey R.P.
        • Kleyman T.R.
        J. Biol. Chem. 2007; 282: 6153-6160
        • Hughey R.P.
        • Bruns J.B.
        • Kinlough C.L.
        • Harkleroad K.L.
        • Tong Q.
        • Carattino M.D.
        • Johnson J.P.
        • Stockand J.D.
        • Kleyman T.R.
        J. Biol. Chem. 2004; 279: 18111-18114
        • Passero C.J.
        • Carattino M.D.
        • Kashlan O.B.
        • Myerburg M.M.
        • Hughey R.P.
        • Kleyman T.R.
        Am. J. Physiol. Renal Physiol. 2010; 299: F854-F861
        • Carattino M.D.
        • Passero C.J.
        • Steren C.A.
        • Maarouf A.B.
        • Pilewski J.M.
        • Myerburg M.M.
        • Hughey R.P.
        • Kleyman T.R.
        Am. J. Physiol. Renal Physiol. 2008; 294: F47-F52
        • Carattino M.D.
        • Sheng S.
        • Bruns J.B.
        • Pilewski J.M.
        • Hughey R.P.
        • Kleyman T.R.
        J. Biol. Chem. 2006; 281: 18901-18907
        • Pinheiro J.C.
        • Bates D.M.
        Chambers J. Eddy W. Härdle W. Sheather S. Tierney L. Mixed-Effects Models in S and S-PLUS. Springer-Verlag, New York2000: 271-414
        • Sheng S.
        • Carattino M.D.
        • Bruns J.B.
        • Hughey R.P.
        • Kleyman T.R.
        Am. J. Physiol. Renal Physiol. 2006; 290: F1488-F1496
        • Condliffe S.B.
        • Zhang H.
        • Frizzell R.A.
        J. Biol. Chem. 2004; 279: 10085-10092
        • Carattino M.D.
        • Hughey R.P.
        • Kleyman T.R.
        J. Biol. Chem. 2008; 283: 25290-25295
        • Maarouf A.B.
        • Sheng N.
        • Chen J.
        • Winarski K.L.
        • Okumura S.
        • Carattino M.D.
        • Boyd C.R.
        • Kleyman T.R.
        • Sheng S.
        J. Biol. Chem. 2009; 284: 7756-7765
        • Horovitz A.
        Fold. Des. 1996; 1: R121-R126
        • Winarski K.L.
        • Sheng N.
        • Chen J.
        • Kleyman T.R.
        • Sheng S.
        J. Biol. Chem. 2010; 285: 26088-26096
        • Clark E.B.
        • Jovov B.
        • Rooj A.K.
        • Fuller C.M.
        • Benos D.J.
        J. Biol. Chem. 2010; 285: 27130-27143
        • Kleyman T.R.
        • Carattino M.D.
        • Hughey R.P.
        J. Biol. Chem. 2009; 284: 20447-20451
        • Bize V.
        • Horisberger J.D.
        Am. J. Physiol. Renal Physiol. 2007; 293: F1137-F1146