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Inhibitory Tract Traps the Epithelial Na+ Channel in a Low Activity Conformation*

  • Ossama B. Kashlan
    Affiliations
    Department of Medicine, Renal-Electrolyte Division and University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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  • Brandon M. Blobner
    Affiliations
    Department of Medicine, Renal-Electrolyte Division and University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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  • Zachary Zuzek
    Affiliations
    Department of Medicine, Renal-Electrolyte Division and University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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  • Marcelo D. Carattino
    Affiliations
    Department of Medicine, Renal-Electrolyte Division and University of Pittsburgh, Pittsburgh, Pennsylvania 15261

    Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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  • Thomas R. Kleyman
    Correspondence
    To whom correspondence should be addressed: Dept. of Medicine, Renal-Electrolyte Div., Dept. of Cell Biology and Physiology, University of Pittsburgh, A919 Scaife, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-647-3121; Fax: 412-648-9166;
    Affiliations
    Department of Medicine, Renal-Electrolyte Division and University of Pittsburgh, Pittsburgh, Pennsylvania 15261

    Department of Cell Biology and Physiology, 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 DK078734 (to O. B. K.), DK065161 (to T. R. K.), DK084060 (to M. D. C.), and DK079307 (the Pittsburgh Center for Kidney Research).
    This article contains supplemental Table 1.
Open AccessPublished:April 17, 2012DOI:https://doi.org/10.1074/jbc.M112.358218
      Proteolysis plays an important role in the maturation and activation of epithelial Na+ channels (ENaCs). Non-cleaved channels are inactive at high extracellular Na+ concentrations and fully cleaved channels are constitutively active. Cleavage of the α and γ subunits at multiple sites activates the channel through the release of imbedded inhibitory tracts. Peptides derived from these released tracts are also inhibitory, likely through binding at the inhibitory tract sites. We recently reported a model of the α subunit. We have now cross-linked Cys derivatives of the inhibitory peptide to the channel, using our model to predict sites at a domain interface of the α subunit that is in proximity to the N terminus of the peptide. Furthermore, peptide inhibition was mimicked in the absence of peptide by cross-linking the channel across the domain interface. Our results suggest a dynamic domain interface that can be exploited by inhibitory peptides and provides a mechanism for peptide inhibition and proteolytic activation.

      Introduction

      Na+ is the predominant cation in the extracellular fluids. Na+ balance and extracellular fluid volume homeostasis are linked directly. The epithelial Na+ channel (ENaC)
      The abbreviations used are: ENaC
      epithelial Na+ channel
      Po
      open probability
      MTS
      methanethiosulfonate
      MTSET
      2-(trimethylammonium)ethyl methanethiosulfonate
      MTSES
      2-sulfonatoethyl methanethiosulfonate
      ANOVA
      analysis of variance
      N-Cys
      CLPHPLQRL
      C-Cys
      LPHPLQRLC.
      is crucial to extracellular fluid volume homeostasis through its role in transepithelial Na+ transport in the aldosterone-sensitive distal nephron (
      • Kashlan O.B.
      • Kleyman T.R.
      ENaC structure and function in the wake of a resolved structure of a family member.
      ,
      • Bhalla V.
      • Hallows K.R.
      Mechanisms of ENaC regulation and clinical implications.
      ). ENaC also participates in the regulation of airway surface liquid volume by facilitating Na+ transport across airway epithelia. It is a member of the ENaC/degenerin family of ion channels, which are gated by ligands and/or mechanical stimuli, and share several structural and functional features.
      Rare among ion channels, ENaC is activated by proteolysis (
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
      ). This processing event transitions channels to a high open probability (Po) state and tailors channel function to its biological role in bulk Na+ transport. We have suggested previously that proteolysis provided a mechanism that allowed ENaCs to evolve from other members of the ENaC/degenerin family that reside primarily in the closed state and transiently open 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.
      ). Cleavage likely occurs during channel maturation by proteases in the biosynthetic pathway, as well as at the cell surface (
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
      ). ENaC proteolysis has been suggested to play a role in disorders such as nephrotic syndrome and cystic fibrosis, where concentrations of specific protease are elevated in the extracellular milieu (
      • Passero C.J.
      • Mueller G.M.
      • Rondon-Berrios H.
      • Tofovic S.P.
      • Hughey R.P.
      • Kleyman T.R.
      Plasmin activates epithelial Na+ channels by cleaving the γ subunit.
      ,
      • Caldwell R.A.
      • Boucher R.C.
      • Stutts M.J.
      Neutrophil elastase activates near-silent epithelial Na+-channels and increases airway epithelial Na+-transport.
      ). Activation requires cleavage at multiple sites within the α and/or γ subunits with the liberation of imbedded inhibitory tracts (
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
      ,
      • Carattino M.D.
      • Sheng S.
      • Bruns J.B.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its a subunit.
      ,
      • Bruns J.B.
      • Carattino M.D.
      • Sheng S.
      • Maarouf A.B.
      • Weisz O.A.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the γ subunit.
      ). Crucially, it is the release of inhibitory tracts and not cleavage itself that activates ENaC. Consequently, peptides that correspond to the released fragments are inhibitory (
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
      ,
      • Carattino M.D.
      • Sheng S.
      • Bruns J.B.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its a subunit.
      ,
      • Bruns J.B.
      • Carattino M.D.
      • Sheng S.
      • Maarouf A.B.
      • Weisz O.A.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      Epithelial Na+ channels are fully activated by furin- and prostasin-dependent release of an inhibitory peptide from the γ subunit.
      ,
      • Carattino M.D.
      • Passero C.J.
      • Steren C.A.
      • Maarouf A.B.
      • Pilewski J.M.
      • Myerburg M.M.
      • Hughey R.P.
      • Kleyman T.R.
      Defining an inhibitory domain in the α subunit of the epithelial sodium channel.
      ,
      • Passero C.J.
      • Carattino M.D.
      • Kashlan O.B.
      • Myerburg M.M.
      • Hughey R.P.
      • Kleyman T.R.
      Defining an inhibitory domain in the γ subunit of the epithelial sodium channel.
      ). We recently reported that introduction of Trp mutations at a large number of sites in the α subunit reduced the inhibition of channel activity by the α subunit-derived 8-mer peptide, Ac-LPHPLQRL-amide (
      • Kashlan O.B.
      • Boyd C.R.
      • Argyropoulos C.
      • Okumura S.
      • Hughey R.P.
      • Grabe M.
      • Kleyman T.R.
      Allosteric Inhibition of the Epithelial Na+ Channel (ENaC) through Peptide Binding at Peripheral Finger and Thumb Domains.
      ). A structural model of the α subunit extracellular region was constructed based on these data and on homology to ASIC1 (
      • 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.
      ), the only member of the ENaC/degenerin family whose structure has been resolved (
      • Jasti J.
      • Furukawa H.
      • Gonzales E.B.
      • Gouaux E.
      Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH.
      ,
      • Gonzales E.B.
      • Kawate T.
      • Gouaux E.
      Pore architecture and ion sites in acid-sensing ion channels and P2X receptors.
      ). ASIC1 is homotrimeric, and the extracellular region of each subunit has three peripheral helical domains (termed the thumb, finger, and knuckle) surrounding two central β sheet domains (termed palm and β-ball) that form the core of the structure. Our model placed the bound peptide at the finger-thumb domain interface, suggesting that this domain interface changes conformation upon gating. Motions around this interface were correlated with twisting motions in the pore in normal mode analysis calculations performed on both ASIC1 (
      • Yang H.
      • Yu Y.
      • Li W.G.
      • Yu F.
      • Cao H.
      • Xu T.L.
      • Jiang H.
      Inherent dynamics of the acid-sensing ion channel 1 correlates with the gating mechanism.
      ) and our α ENaC model (
      • 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.
      ).
      To test our model of inhibitory peptide binding, we attempted to cross-link Cys derivatives of the peptide to the channel using bifunctional methanethiosulfonate (MTS) compounds. We found that sites at the finger-thumb interface crosslink to the N terminus of the peptide but not the C terminus. We also found sites in the finger domain further from the finger-thumb interface that cross-link to the C terminus of the peptide but not the N terminus. In probing the mechanism of peptide inhibition, we found that cross-linking adjacent sites at the thumb-finger interface trapped the channel in a low activity state. Our data suggest a dynamic finger-thumb interface. Narrowing the interface transitions the channel to a low activity state, providing a mechanism for peptide inhibition and proteolytic ENaC activation.

      DISCUSSION

      Our α ENaC model places the N terminus of the inhibitory 8-mer peptide at the finger-thumb interface, and its C terminus near the beginning of helix α2, far from the finger-thumb interface (
      • 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.
      ). Cross-linking data are clearly consistent with this model of inhibitory peptide binding. We hypothesized that the peptide draws the finger and thumb together in the peptide bound state, which is inhibitory. Peptide release frees the finger and thumb to move, allowing the channel to more readily assume an open conformation. This idea is supported by our demonstration that cross-linking D171C and Y474C across the finger-thumb interface reduces channel activity. These sites were selected because they each cross-linked with the N-Cys peptide and were in close proximity in our α ENaC model. The inhibitory effect of cross-linking D171C and Y474C demonstrated a length requirement, suggesting that linkers with three or fewer carbons sufficiently constrained the finger-thumb interface to favor the closed channel conformation. Longer linkers either did not cross-link or allowed sufficient mobility at the interface so as to have little effect. We also reproduced the cross-linking effect by placing opposing charges at this interface. Taken together, these data are the first experimental evidence of movement between the finger and thumb domains of ENaC and show that such rearrangements affect channel gating. The stabilization of a specific conformation at a dynamic domain interface provides a mechanism for the allosteric inhibition of ENaC by peptides. We propose that proteolytic ENaC activation functions in an analogous manner by releasing a constraint at the finger-thumb interface.
      In all of our experiments designed to constrain movements between the α4–α5 loop of the thumb domain and helix α1 of the finger domain, we never observed >50% inhibition despite saturating the effect of the MTS reagent. We draw two important conclusions from this observation. First, the gate does not change its conformation in a lockstep mechanism with the finger-thumb interface. Cross-linked channels still transition between closed and open states but likely exhibit a lower Po than their non-cross-linked counterparts. Second, the 26-mer and 8-mer peptides derived from the 206–231 inhibitory tract also inhibit ENaC through additional mechanisms. We conclude this based on the fact that each peptide can achieve 80–90% inhibition at saturation. Our model postulates extensive interactions between the peptide and other parts of the finger domain, which suggests additional structures have a role in peptide inhibition.
      Our data link dynamic changes at the finger-thumb interface to functional changes at the channel gate. Although we were able to inhibit the channel by exploiting this interface, we were not able to stimulate the channel. We attempted to stimulate ENaC currents by introducing like charges at the interface. Modification of Y474C channels with MTSES led to reduced channel activity, which was an unexpected result. Furthermore, DTT did not reverse the effect of MTSES, in contrast to its effect on MTSET-modified Y474C channels. It is possible that MTSES modification led to a change in the conformation of the α4–α5 loop that prevented DTT access to the modified Cys at position 474. Alternatively, MTSES modification led to misfolding of the α4–α5 loop that could not be reversed even after MTSES modification was reversed by DTT. We speculate that rather than pushing helix α1 and the α4–α5 loop apart, MTSES modification of Y474C led to a non-reversible thumb loop conformational change that reduced channel currents.
      The thumb domains of the ENaC/degenerin family of ion channels are poorly conserved yet share two important features: two predicted helices and 10 absolutely conserved Cys that form a five-disulfide bond ladder (
      • Jasti J.
      • Furukawa H.
      • Gonzales E.B.
      • Gouaux E.
      Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH.
      ,
      • Sheng S.
      • Maarouf A.B.
      • Bruns J.B.
      • Hughey R.P.
      • Kleyman T.R.
      Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition.
      ). These hallmark features of ENaC/degenerin channels suggest that the antiparallel arrangement of the two thumb helices in ASIC1 is widely conserved. In the ASIC1 structure, the thumb connects to the palm near the opening of the pore and extends up from the membrane toward the finger. The thumb also interacts with the β-ball and finger of the same subunit and the palm of a neighboring subunit. Our data suggests that the 8-mer inhibitory peptide exploits the finger-thumb interface to stabilize the channel in the closed conformation. Recent data suggest that Cl, which inhibits ENaC, binds at the thumb-palm intersubunit interface (
      • Collier D.M.
      • Snyder P.M.
      Extracellular chloride regulates the epithelial sodium channel.
      ,
      • Collier D.M.
      • Snyder P.M.
      Identification of epithelial Na+ channel (ENaC) intersubunit Cl inhibitory residues suggests a trimeric αγβ channel architecture.
      ). It remains to be determined whether the relative finger-thumb movements also involve the thumb-palm intersubunit interface. Furthermore, it is unclear whether inhibitory peptides, Cl, and other extracellular allosteric inhibitors (e.g. Na+) recruit the same allosteric machinery but use different sites to trigger conformational changes. The rigidity implied by the presence of five disulfide bonds in the thumb suggests that movement at any thumb interdomain interface will induce movement at other thumb interdomain interfaces. We reported recently that mutations in the thumb near the outer mouth of the pore modulated the efficacy of multiple extracellular stimuli, suggesting mechanistic convergence (
      • 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.
      ). The remarkably similar kinetics of peptide, Cl, and Na+ inhibition hint at a common rate-limiting step (
      • Kashlan O.B.
      • Boyd C.R.
      • Argyropoulos C.
      • Okumura S.
      • Hughey R.P.
      • Grabe M.
      • Kleyman T.R.
      Allosteric Inhibition of the Epithelial Na+ Channel (ENaC) through Peptide Binding at Peripheral Finger and Thumb Domains.
      ,
      • Collier D.M.
      • Snyder P.M.
      Extracellular chloride regulates the epithelial sodium channel.
      ,
      • Sheng S.
      • Bruns J.B.
      • Kleyman T.R.
      Extracellular histidine residues crucial for Na+ self-inhibition of epithelial Na+ channels.
      ,
      • Chraïbi A.
      • Horisberger J.D.
      Na self inhibition of human epithelial Na channel: Temperature dependence and effect of extracellular proteases.
      ). As ENaC/degenerin channels have evolved to respond to numerous extracellular stimuli, the basic architecture of the thumb domain seems to have been preserved. In this work, we found evidence for movement at a finger-thumb interface where an inhibitory peptide binds. Influencing interdomain interfaces that involve the thumb may be a general mechanism for extracellular ligand gating among ENaC/degenerin channels.

      Acknowledgments

      We thank Michael Grabe for helpful discussions.

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