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Regulation of Epithelial Sodium Channel Trafficking by Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9)*

  • Vikas Sharotri
    Affiliations
    Departments of Internal Medicine and Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242
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  • Daniel M. Collier
    Affiliations
    Departments of Internal Medicine and Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242
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  • Diane R. Olson
    Affiliations
    Departments of Internal Medicine and Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242
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  • Ruifeng Zhou
    Affiliations
    Departments of Internal Medicine and Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242
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  • Peter M. Snyder
    Correspondence
    To whom correspondence should be addressed: 371 EMRB, University of Iowa, Iowa City, IA 52242
    Affiliations
    Departments of Internal Medicine and Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant HL058812 (to P. M. S.). This work was also supported by fellowship grants from the American Heart Association (to V. S. and D. M. C.).
      The epithelial Na+ channel (ENaC) is critical for Na+ homeostasis and blood pressure control. Defects in its regulation cause inherited forms of hypertension and hypotension. Previous work found that ENaC gating is regulated by proteases through cleavage of the extracellular domains of the α and γ subunits. Here we tested the hypothesis that ENaC is regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), a protease that modulates the risk of cardiovascular disease. PCSK9 reduced ENaC current in Xenopus oocytes and in epithelia. This occurred through a decrease in ENaC protein at the cell surface and in the total cellular pool, an effect that did not require the catalytic activity of PCSK9. PCSK9 interacted with all three ENaC subunits and decreased their trafficking to the cell surface by increasing proteasomal degradation. In contrast to its previously reported effects on the LDL receptor, PCSK9 did not alter ENaC endocytosis or degradation of the pool of ENaC at the cell surface. These results support a role for PCSK9 in the regulation of ENaC trafficking in the biosynthetic pathway, likely by increasing endoplasmic reticulum-associated degradation. By reducing ENaC channel number, PCSK9 could modulate epithelial Na+ absorption, a major contributor to blood pressure control.

      Introduction

      The epithelial Na+ channel (ENaC)
      The abbreviations used are: ENaC
      epithelial Na+ channel
      PCSK9
      proprotein convertase subtilisin/kexin type 9
      LDLR
      LDL receptor
      FRT
      Fischer rat thyroid
      MTSET
      [2-(trimethylammonium)ethyl]methanethiosulfonate bromide.
      plays an important role in absorption of Na+ across epithelia, including the kidney, collecting duct and connecting tubule, lung, distal colon, and sweat duct (reviewed in Refs.
      • Schild L.
      The epithelial sodium channel. From molecule to disease.
      ,
      • Snyder P.M.
      Minireview. Regulation of epithelial Na+ channel trafficking.
      ). A heterotrimer composed of three homologous subunits (α, β, and γ), ENaC is expressed at the apical membrane where it forms a pathway for Na+ to enter the cell. Na+ leaves the cell at the basolateral membrane via the Na+-K+-ATPase, which completes the pathway for Na+ absorption. This process is critical to control extracellular volume and to maintain the composition and quantity of epithelial surface liquid. This is illustrated by several diseases. For example, ENaC mutations that slow its retrieval from the cell surface cause an inherited form of hypertension (Liddle's syndrome), resulting from excessive renal Na+ absorption (
      • Snyder P.M.
      • Price M.P.
      • McDonald F.J.
      • Adams C.M.
      • Volk K.A.
      • Zeiher B.G.
      • Stokes J.B.
      • Welsh M.J.
      Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel.
      ,
      • Firsov D.
      • Schild L.
      • Gautschi I.
      • Mérillat A.M.
      • Schneeberger E.
      • Rossier B.C.
      Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome. A quantitative approach.
      ,
      • Knight K.K.
      • Olson D.R.
      • Zhou R.
      • Snyder P.M.
      Liddle's syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage.
      ). Defects in ENaC regulation are responsible for most of the known genetic forms of hypertension (
      • Lifton R.P.
      Molecular genetics of human blood pressure variation.
      ). Conversely, loss of function mutations cause pseudohypoaldosteronism type I, a disorder of renal Na+ wasting (
      • Chang S.S.
      • Grunder S.
      • Hanukoglu A.
      • Rösler A.
      • Mathew P.M.
      • Hanukoglu I.
      • Schild L.
      • Lu Y.
      • Shimkets R.A.
      • Nelson-Williams C.
      • Rossier B.C.
      • Lifton R.P.
      Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1.
      ). In the lung, defects in ENaC activity cause pulmonary edema and may contribute to the pathogenesis of cystic fibrosis (
      • Mall M.
      • Grubb B.R.
      • Harkema J.R.
      • O'Neal W.K.
      • Boucher R.C.
      Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice.
      ).
      Previous work indicates that ENaC is regulated by serine proteases (reviewed in Ref.
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
      ). Furin, a member of the proprotein convertase family, cleaves the extracellular domain of αENaC at basic motifs, removing a 26-amino acid fragment (
      • Hughey R.P.
      • Mueller G.M.
      • Bruns J.B.
      • Kinlough C.L.
      • Poland P.A.
      • Harkleroad K.L.
      • Carattino M.D.
      • Kleyman T.R.
      Maturation of the epithelial Na+ channel involves proteolytic processing of the α and γ subunits.
      ). In γENaC, furin cleaves the extracellular domain at a single site and a second, more distal site is cleaved by additional proteases (e.g. CAP1/prostasin, plasmin, elastase), releasing a fragment of ∼43 amino acids (
      • Vallet V.
      • Chraibi A.
      • Gaeggeler H.P.
      • Horisberger J.D.
      • Rossier B.C.
      An epithelial serine protease activates the amiloride-sensitive sodium channel.
      ,
      • Adebamiro A.
      • Cheng Y.
      • Rao U.S.
      • Danahay H.
      • Bridges R.J.
      A segment of γ ENaC mediates elastase activation of Na+ transport.
      ,
      • 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.
      ,
      • Caldwell R.A.
      • Boucher R.C.
      • Stutts M.J.
      Neutrophil elastase activates near-silent epithelial Na+ channels and increases airway epithelial Na+ transport.
      ,
      • 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.
      ). Proteolytic cleavage of α- and γENaC converts quiescent channels into active Na+-conducting channels. This activation occurs by relieving the channel from inhibition by extracellular Na+ (“Na+ self-inhibition”) (
      • Chraïbi A.
      • Horisberger J.D.
      Na self-inhibition of human epithelial Na channel. Temperature dependence and effect of extracellular proteases.
      ). Proteolytic cleavage of ENaC is a regulated process. For example, cleavage is inhibited by increased intracellular Na+, providing a negative feedback mechanism to regulate Na+ absorption (
      • Knight K.K.
      • Wentzlaff D.M.
      • Snyder P.M.
      Intracellular sodium regulates proteolytic activation of the epithelial sodium channel.
      ). Conversely, cleavage is enhanced by Na+ depletion and aldosterone infusion (
      • Frindt G.
      • Masilamani S.
      • Knepper M.A.
      • Palmer L.G.
      Activation of epithelial Na channels during short-term Na deprivation.
      ,
      • Masilamani S.
      • Kim G.H.
      • Mitchell C.
      • Wade J.B.
      • Knepper M.A.
      Aldosterone-mediated regulation of ENaC α, β, and γ subunit proteins in rat kidney.
      ). Cleavage is also disrupted in pathological states. In Liddle's syndrome, cleavage is increased, likely through prolonged exposure of ENaC to proteases present at the cell surface (
      • Knight K.K.
      • Olson D.R.
      • Zhou R.
      • Snyder P.M.
      Liddle's syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage.
      ). There is also evidence to suggest that ENaC cleavage is increased in nephrotic syndrome (
      • 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.
      ,
      • Svenningsen P.
      • Bistrup C.
      • Friis U.G.
      • Bertog M.
      • Haerteis S.
      • Krueger B.
      • Stubbe J.
      • Jensen O.N.
      • Thiesson H.C.
      • Uhrenholt T.R.
      • Jespersen B.
      • Jensen B.L.
      • Korbmacher C.
      • Skøtt O.
      Plasmin in nephrotic urine activates the epithelial sodium channel.
      ) and cystic fibrosis (
      • Myerburg M.M.
      • Butterworth M.B.
      • McKenna E.E.
      • Peters K.W.
      • Frizzell R.A.
      • Kleyman T.R.
      • Pilewski J.M.
      Airway surface liquid volume regulates ENaC by altering the serine protease-protease inhibitor balance. A mechanism for sodium hyperabsorption in cystic fibrosis.
      ,
      • Tarran R.
      • Trout L.
      • Donaldson S.H.
      • Boucher R.C.
      Soluble mediators, not cilia, determine airway surface liquid volume in normal and cystic fibrosis superficial airway epithelia.
      ).
      Because proteolytic cleavage modulates ENaC gating, there has been considerable interest in identifying additional proteases that regulate ENaC. The proprotein convertase family has nine members, including furin (
      • Seidah N.G.
      • Mayer G.
      • Zaid A.
      • Rousselet E.
      • Nassoury N.
      • Poirier S.
      • Essalmani R.
      • Prat A.
      The activation and physiological functions of the proprotein convertases.
      ). In this work, we investigated a potential role for another member of this family, proprotein convertase subtilisin/kexin type 9 (PCSK9) (
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1). Liver regeneration and neuronal differentiation.
      ). Consistent with a potential role in ENaC regulation, PCSK9 is expressed in the kidney and lung (
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1). Liver regeneration and neuronal differentiation.
      ). It is synthesized as a 72-kDa immature precursor that undergoes autocatalytic cleavage in the endoplasmic reticulum to generate a 63-kDa mature protein (
      • Benjannet S.
      • Rhainds D.
      • Essalmani R.
      • Mayne J.
      • Wickham L.
      • Jin W.
      • Asselin M.C.
      • Hamelin J.
      • Varret M.
      • Allard D.
      • Trillard M.
      • Abifadel M.
      • Tebon A.
      • Attie A.D.
      • Rader D.J.
      • Boileau C.
      • Brissette L.
      • Chrétien M.
      • Prat A.
      • Seidah N.G.
      NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.
      ). The cleaved N-terminal fragment remains associated with the mature protein and is necessary for its secretion, allowing it to circulate in the blood (
      • Lagace T.A.
      • Curtis D.E.
      • Garuti R.
      • McNutt M.C.
      • Park S.W.
      • Prather H.B.
      • Anderson N.N.
      • Ho Y.K.
      • Hammer R.E.
      • Horton J.D.
      Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.
      ).
      Previous work has focused on the role of PCSK9 in the regulation of the LDL receptor (LDLR). By reducing expression of the LDLR at the cell surface, PCSK9 increases serum levels of LDL cholesterol (
      • Benjannet S.
      • Rhainds D.
      • Essalmani R.
      • Mayne J.
      • Wickham L.
      • Jin W.
      • Asselin M.C.
      • Hamelin J.
      • Varret M.
      • Allard D.
      • Trillard M.
      • Abifadel M.
      • Tebon A.
      • Attie A.D.
      • Rader D.J.
      • Boileau C.
      • Brissette L.
      • Chrétien M.
      • Prat A.
      • Seidah N.G.
      NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.
      ,
      • Maxwell K.N.
      • Fisher E.A.
      • Breslow J.L.
      Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment.
      ,
      • Park S.W.
      • Moon Y.A.
      • Horton J.D.
      Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver.
      ). Rare gain-of-function PCSK9 mutations cause hypercholesterolemia and increase the risk of coronary heart disease, whereas loss-of-function mutations cause hypocholesterolemia and protect against heart disease (
      • Horton J.D.
      • Cohen J.C.
      • Hobbs H.H.
      Molecular biology of PCSK9. Its role in LDL metabolism.
      ,
      • Seidah N.G.
      • Prat A.
      The proprotein convertases are potential targets in the treatment of dyslipidemia.
      ,
      • Maxwell K.N.
      • Breslow J.L.
      Proprotein convertase subtilisin kexin 9. The third locus implicated in autosomal dominant hypercholesterolemia.
      ,
      • Cohen J.C.
      • Boerwinkle E.
      • Mosley Jr., T.H.
      • Hobbs H.H.
      Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.
      ,
      • McNutt M.C.
      • Lagace T.A.
      • Horton J.D.
      Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells.
      ). The mechanisms by which PCSK9 alters LDLR surface expression are not completely understood. Secreted PCSK9 (or recombinant PCSK9 added to the extracellular medium) binds to the LDLR and undergoes endocytosis (
      • Lagace T.A.
      • Curtis D.E.
      • Garuti R.
      • McNutt M.C.
      • Park S.W.
      • Prather H.B.
      • Anderson N.N.
      • Ho Y.K.
      • Hammer R.E.
      • Horton J.D.
      Secreted PCSK9 decreases the number of LDL receptors in hepatocytes and in livers of parabiotic mice.
      ,
      • Maxwell K.N.
      • Fisher E.A.
      • Breslow J.L.
      Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment.
      ,
      • Nassoury N.
      • Blasiole D.A.
      • Tebon Oler A.
      • Benjannet S.
      • Hamelin J.
      • Poupon V.
      • McPherson P.S.
      • Attie A.D.
      • Prat A.
      • Seidah N.G.
      The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR.
      ,
      • Zhang D.W.
      • Lagace T.A.
      • Garuti R.
      • Zhao Z.
      • McDonald M.
      • Horton J.D.
      • Cohen J.C.
      • Hobbs H.H.
      Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation.
      ,
      • Kwon H.J.
      • Lagace T.A.
      • McNutt M.C.
      • Horton J.D.
      • Deisenhofer J.
      Molecular basis for LDL receptor recognition by PCSK9.
      ). In the endocytic pathway, PCSK9 increases lysosomal degradation of the LDLR. Although secreted PCSK9 regulates LDLR trafficking, additional evidence suggests that PCSK9 may also induce LDLR degradation through an intracellular route (
      • Poirier S.
      • Mayer G.
      • Poupon V.
      • McPherson P.S.
      • Desjardins R.
      • Ly K.
      • Asselin M.C.
      • Day R.
      • Duclos F.J.
      • Witmer M.
      • Parker R.
      • Prat A.
      • Seidah N.G.
      Dissection of the endogenous cellular pathways of PCSK9-induced low density lipoprotein receptor degradation. Evidence for an intracellular route.
      ). Interestingly, although PCSK9 induces degradation of the LDLR, its protease activity is not required (
      • McNutt M.C.
      • Lagace T.A.
      • Horton J.D.
      Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells.
      ,
      • Poirier S.
      • Mayer G.
      • Benjannet S.
      • Bergeron E.
      • Marcinkiewicz J.
      • Nassoury N.
      • Mayer H.
      • Nimpf J.
      • Prat A.
      • Seidah N.G.
      The proprotein convertase PCSK9 induces the degradation of low density lipoprotein receptor (LDLR) and its closest family members VLDLR and ApoER2.
      ). Thus, it has been proposed that PCSK9 regulates the LDLR through a chaperone mechanism, rather than through its function as a protease. Although it seems clear that the PCSK9 regulates the LDLR and two closely related receptors (very low density lipoprotein receptor and apolipoprotein E receptor 2 (
      • Poirier S.
      • Mayer G.
      • Benjannet S.
      • Bergeron E.
      • Marcinkiewicz J.
      • Nassoury N.
      • Mayer H.
      • Nimpf J.
      • Prat A.
      • Seidah N.G.
      The proprotein convertase PCSK9 induces the degradation of low density lipoprotein receptor (LDLR) and its closest family members VLDLR and ApoER2.
      )), additional substrates for PCSK9 have not been identified. Here we show that PCSK9 regulates ENaC and we explore the mechanisms that underlie this regulation.

      EXPERIMENTAL PROCEDURES

       DNA Constructs

      Human αENaC, βENaC, and γENaC were cloned in pMT3 as described previously (
      • McDonald F.J.
      • Snyder P.M.
      • McCray Jr., P.B.
      • Welsh M.J.
      Cloning, expression, and tissue distribution of a human amiloride-sensitive Na+ channel.
      ,
      • McDonald F.J.
      • Price M.P.
      • Snyder P.M.
      • Welsh M.J.
      Cloning and expression of the β and γ subunits of the human epithelial sodium channel.
      ). Mutations were generated by site-directed mutagenesis (QuikChange; Stratagene). αENaC-FLAG, βENaC-FLAG, and γENaC-FLAG were generated by insertion of a FLAG epitope (DYKDDDDK) at the C terminus (
      • Knight K.K.
      • Olson D.R.
      • Zhou R.
      • Snyder P.M.
      Liddle's syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage.
      ,
      • Zhou R.
      • Patel S.V.
      • Snyder P.M.
      Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC.
      ). Human PCSK9-V5 was a generous gift from Nabil Seidah (
      • Seidah N.G.
      • Benjannet S.
      • Wickham L.
      • Marcinkiewicz J.
      • Jasmin S.B.
      • Stifani S.
      • Basak A.
      • Prat A.
      • Chretien M.
      The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1). Liver regeneration and neuronal differentiation.
      ), and Nedd4-2-HA was generated as described (
      • Snyder P.M.
      • Olson D.R.
      • Kabra R.
      • Zhou R.
      • Steines J.C.
      cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4-2.
      ). Mutations were generated (QuikChange, Stratagene) in αENaC (Y644A, R175A, R177A, R178A, R181A, R190A, R192A, R201A, R204A), βENaC (Y620A), and γENaC (Y627A) (G536C) as described previously (
      • Snyder P.M.
      • Price M.P.
      • McDonald F.J.
      • Adams C.M.
      • Volk K.A.
      • Zeiher B.G.
      • Stokes J.B.
      • Welsh M.J.
      Mechanism by which Liddle's syndrome mutations increase activity of a human epithelial Na+ channel.
      ,
      • Snyder P.M.
      • Olson D.R.
      • Bucher D.B.
      A pore segment in DEG/ENaC Na(+) channels.
      ,
      • Kabra R.
      • Knight K.K.
      • Zhou R.
      • Snyder P.M.
      Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels.
      ) and in PCSK9 (S386A). All cDNAs were sequenced in the University of Iowa DNA Core Facility.

       Electrophysiology in Xenopus Oocytes

      Oocytes were harvested from Xenopus laevis females. They were treated for 1 h with 0.75 mg/ml type IV collagenase (Sigma) in Ca2+-free ND-96 (96 mm NaCl, 2 mm KCl, 1 mm MgCl2, 5 mm HEPES (pH 7.4)) and manually defolliculated (
      • 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.
      ,
      • Collier D.M.
      • Snyder P.M.
      Identification of epithelial Na+ channel (ENaC) intersubunit Cl- inhibitory residues suggests a trimeric α γ β channel architecture.
      ). The cell nucleus was injected with cDNAs encoding either human α-, β- (wild-type or S520K), and γENaC (0.2 ng each) or with human ASIC1 (0.6 ng) along with PCSK9 (0–2 ng). Total injected cDNA was held constant using pMT3-SEAP (
      • Swick A.G.
      • Janicot M.
      • Cheneval-Kastelic T.
      • McLenithan J.C.
      • Lane M.D.
      Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes.
      ). Following injection, oocytes were incubated at 18 °C in modified Barth's saline (88 mm NaCl, 1 mm KCl, 0.33 mm Ca(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4, 2.4 mm NaHCO3, 10 mm HEPES, 50 μg/ml gentamicin sulfate, 10 μg/ml sodium penicillin, 10 μg/ml streptomycin sulfate (pH 7.4)) for 20–24 h before electrophysiological recording.
      Oocytes were voltage-clamped at −60 mV, and currents were recorded by two-electrode voltage clamp using an oocyte clamp (OC-725C, Warner Instruments), digitized with a Powerlab interface (ADInstruments), and recorded and analyzed with Chart software (ADInstruments). The cells were bathed in 116 mm NaCl, 2 mm KCl, 0.4 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES (pH 7.4 or 5). The amiloride-sensitive ENaC current was measured by adding 10 μm amiloride to the bathing solution. ASIC1 currents were detected by addition of pH 5 to the bathing solution.

       Electrophysiology in Epithelia

      To test the effect of PCSK9 on ENaC current in epithelia, Fischer rat thyroid (FRT) cells were cultured on permeable filter supports (Millicell PCF, 0.4-μm pore size, 12-mm diameter) in F-12 Coon's medium (Sigma) with 5% fetal calf serum (Sigma), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C. Cells were transfected with α-, β-, and γENaC (0.03 μg each) with or without PCSK9 (0–0.9 μg) using TFX50 (Promega) as described previously (
      • Kabra R.
      • Knight K.K.
      • Zhou R.
      • Snyder P.M.
      Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels.
      ,
      • Snyder P.M.
      Liddle's syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na(+) channel to the cell surface.
      ). Total cDNA was kept constant using GFP cDNA (which does not alter ENaC current). Two days after transfection, the current was measured in Ussing chambers under short-circuit conditions using an EC-825 amplifier (Warner Instruments), digitized with a Powerlab interface (ADInstruments), and recorded and analyzed with Chart software (ADInstruments). The apical and basolateral surfaces were bathed in 135 mm NaCl, 1.2 mm CaCl2, 1.2 mm MgCl2, 2.4 mm K2HPO4, 0.6 mm KH2PO4, and 10 mm HEPES (pH 7.4) at 37 °C. Amiloride (10 μm) was added to the apical solution to quantitate ENaC current.
      For exocytosis experiments, FRT cells were transfected with αENaC, βENaC, and γ536CENaC (0.167 μg each subunit) with PCSK9 or GFP cDNA (0.5 μg) (
      • Snyder P.M.
      • Olson D.R.
      • Bucher D.B.
      A pore segment in DEG/ENaC Na(+) channels.
      ,
      • Snyder P.M.
      Liddle's syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na(+) channel to the cell surface.
      ). Channels at the cell surface were irreversibly blocked by covalent modification of the introduced cysteine with 1 mm [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET). Following removal of MTSET, we measured the rate of current increase to quantitate exocytosis of unblocked channels. Time constants (τ) were determined by fitting the data to single-exponential equations using IGOR Pro 6.01 software.

       Coimmunoprecipitation

      HEK 293T cells were cultured in Dulbecco's modified Eagle's medium. To test for interactions between ENaC and PCSK9, the cells were transfected with αENaC, βENaC, and γENaC (individually or together, 1 μg each) and PCSK9-V5 or GFP (1 or 3 μg) using Lipofectamine2000 (Invitrogen) (
      • Knight K.K.
      • Olson D.R.
      • Zhou R.
      • Snyder P.M.
      Liddle's syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage.
      ,
      • Zhou R.
      • Patel S.V.
      • Snyder P.M.
      Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC.
      ). One ENaC subunit contained a FLAG epitope. Two days after transfection, the cells were lysed in Nonidet P-40 lysis buffer (0.4% sodium deoxycholate, 1% Nonidet P-40, 63 mm EDTA, 50 mm Tris-HCl (pH 8), and protease inhibitor mixture (Sigma)). 500 μg of cellular protein was immunoprecipitated with anti-FLAG M2 affinity gel (Sigma) or anti-V5 antibody (Invitrogen) with immobilized protein A (Pierce) beads overnight at 4 °C. Following SDS-PAGE, ENaC and PCSK9 were detected by immunoblot analysis using anti-FLAG M2 monoclonal antibody-peroxidase conjugate (1:5000, Sigma) at 1:5000 dilution or anti-V5 antibody (1:5000) and enhanced chemiluminescence (ECL Plus, GE Healthcare).

       Biotinylation

      To quantitate ENaC expression at the cell surface, HEK 293 cells expressing α-, β-, and γENaC (FLAG epitope on one subunit) and PCSK9 or GFP were washed with 4 °C PBS containing 1 mm CaCl2 and 1 mm MgCl2 (PBS-CM). Surface proteins were biotinylated with 0.5 mg/ml sulfo-NHS-biotin (Pierce) for 30 min at 4 °C (
      • Knight K.K.
      • Olson D.R.
      • Zhou R.
      • Snyder P.M.
      Liddle's syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage.
      ). Excess biotin was quenched with 100 mm glycine in PBS-CM for 20 min at 4 °C. The cells were lysed in 1% Nonidet P-40, 150 mm NaCl, 50 mm Tris (pH 7.4), and protease inhibitors (Sigma) at 4 °C, and then centrifuged at 14,000 rpm for 10 min to remove insoluble material. Biotinylated proteins were isolated with NeutrAvidin-agarose beads (Pierce) overnight at 4 °C. Following extensive washing, biotinylated proteins were eluted with SDS sample buffer (4% SDS, 100 mm dithiothreitol, 20% glycerol, and 100 mm Tris-Cl (pH 6.8)) and separated by SDS-PAGE. Biotinylated ENaC and ENaC in the total cellular lysate were detected by immunoblot using anti-FLAG M2-peroxidase-conjugated antibody (1:5000, Sigma) and enhanced chemiluminescence (ECL Plus, GE Healthcare) and quantitated by densitometry (ImageJ) using non-saturated exposures.

       Degradation

      To measure the rate of ENaC degradation, HEK 293 cells transfected with αENaC-FLAG, βENaC, and γENaC with PCSK9 or GFP were incubated with cycloheximide (10 μg/ml) for 0–120 min. Remaining αENaC-FLAG at each time point was detected by immunoblot (anti-FLAG M2-peroxidase-conjugated antibody) and quantitated by densitometry. To identify the location of degradation, cells were treated with 10 μm N-acetyl-Leu-Leu-norleucinal or 5 mm NH4Cl.
      To measure the rate of degradation of the cell surface fraction of ENaC, HEK 293 cells transfected with αENaC-FLAG, βENaC, and γENaC with PCSK9 or GFP were biotinylated on ice and then incubated at 37 °C for 0–120 min (
      • Knight K.K.
      • Olson D.R.
      • Zhou R.
      • Snyder P.M.
      Liddle's syndrome mutations increase Na+ transport through dual effects on epithelial Na+ channel surface expression and proteolytic cleavage.
      ,
      • Zhou R.
      • Patel S.V.
      • Snyder P.M.
      Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC.
      ). Biotinylated αENaC-FLAG was isolated using NeutrAvidin-agarose, detected by immunoblot (anti-FLAG M2-peroxidase-conjugated antibody), and quantitated by densitometry.

       Endocytosis

      To measure the rate of ENaC endocytosis, we used a previously described αENaC construct (αCl-2) in which multiple arginines were simultaneously mutated to prevent proteolytic cleavage by furin but to retain the ability to be cleaved by trypsin (R175A, R177A, R178A, R181A, R190A, R192A, R201A, and R204A) (
      • Kabra R.
      • Knight K.K.
      • Zhou R.
      • Snyder P.M.
      Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels.
      ). HEK 293 cells were transfected with αCl-2 ENaC-FLAG, βENaC, and γENaC with PCSK9 or GFP were incubated with trypsin (5 μg/ml) for 5 min at 37 °C to generate a pool of cleaved channels 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.
      )(
      • Kabra R.
      • Knight K.K.
      • Zhou R.
      • Snyder P.M.
      Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels.
      ). The cells were washed three times with cold PBS-CM to remove trypsin, incubated at 37 °C for 0–60 min to allow endocytosis of cleaved channels, and then placed on ice. Cleaved channels remaining at the cell surface were labeled with biotin, isolated with NeutrAvidin-agarose, detected by immunoblot analysis (anti-FLAG M2-peroxidase-conjugated antibody), and quantitated by densitometry.

      RESULTS

       PCSK9 Inhibits ENaC

      We tested the effect of PCSK9 on ENaC current utilizing two expression systems. First, we injected Xenopus oocytes with α-, β-, and γENaC cDNA to generate amiloride-sensitive Na+ currents (Fig. 1A). We found that coexpression of PCSK9 decreased the Na+ current in a dose-dependent manner (Fig. 1, A and B).
      Figure thumbnail gr1
      FIGURE 1PCSK9 inhibits ENaC current. A and B, Xenopus oocytes were nuclear-injected with cDNAs encoding human α-, β-, and γENaC (0.2 ng each) and PCSK9 (0–2 ng). A, representative current traces (0.2 ng PCSK9). 10 μm amiloride (Amil) was added to the bathing solution as indicated by the black bar. B, summary plot of amiloride-sensitive current versus amount of injected PCSK9 cDNA (mean ± S.E. relative to 0 PCSK9 group, n = 5–26). C and D, FRT epithelia were transfected with α-, β-, and γENaC (0.03 μg each) and PCSK9 (0–0.9 μg). C, representative short-circuit current traces. 10 μm amiloride was added to the apical bathing solution as indicated by the black bar. D, summary plot of amiloride-sensitive current versus the amount of transfected PCSK9 (mean ± S.E. relative to 0 PCSK9 group, n = 9–12). E and F, Xenopus oocytes were nuclear-injected with cDNAs encoding human ASIC1 (0.6 ng) and PCSK9 or control plasmid (0.8 ng). E, representative current traces. The bath was perfused with pH 5 solution as indicated by the black bar. F, summary plot of proton-activated current (mean ± S.E.; n = 9; *, p < 0.03).
      As a second strategy, we tested the effect of PCSK9 on the ENaC current in epithelia. Transfection of FRT epithelia with α-, β-, and γENaC resulted in amiloride-sensitive short-circuit currents (Fig. 1C). Cotransfection with PCSK9 produced a dose-dependent decrease in ENaC current (Fig. 1, C and D), similar to our results in oocytes. Thus, PCSK9 inhibited ENaC in two independent experimental systems.
      We also tested the effect of PCSK9 on a related DEG/ENaC channel, ASIC1. PCSK9 reduced the proton-activated ASIC1 current by 24% in Xenopus oocytes (Fig. 1, E and F), less than its effect on ENaC.

       PCSK9 Interacts with ENaC

      To begin to investigate the mechanism by which PCSK9 inhibits ENaC current, we tested whether PCSK9 and ENaC interact with one another. In Fig. 2A, we transfected HEK 293 cells with α-, β-, and γENaC (one of the subunits contained a FLAG epitope) along with PCSK9 (V5 epitope) and examined protein interactions using a coimmunoprecipitation assay. When we immunoprecipitated αENaC, we detected coprecipitated PCSK9 in cells cotransfected with ENaC and PCSK9 but not in cells transfected individually with either ENaC or PCSK9 (Fig. 2A, first panel). Likewise, we detected PCSK9 when we immunoprecipitated β- or γENaC (Fig. 2A, first panel). There are two forms of PCSK9, full-length pro-PCSK9 (72 kDa) and autocatalytically cleaved PCSK9 (63 kDa) (Fig. 2A, third panel) (
      • Benjannet S.
      • Rhainds D.
      • Essalmani R.
      • Mayne J.
      • Wickham L.
      • Jin W.
      • Asselin M.C.
      • Hamelin J.
      • Varret M.
      • Allard D.
      • Trillard M.
      • Abifadel M.
      • Tebon A.
      • Attie A.D.
      • Rader D.J.
      • Boileau C.
      • Brissette L.
      • Chrétien M.
      • Prat A.
      • Seidah N.G.
      NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.
      ). ENaC selectively coprecipitated the pro-PCSK9 form (Fig. 2A, first panel). Using a reciprocal strategy we found that α-, β-, and γENaC each coprecipitated when we immunoprecipitated PCSK9 (Fig. 2A, second panel). In the immunoblot analysis, we observed two bands for α- and γENaC, which correspond to the full-length (immature) and proteolytically cleaved (mature) forms, respectively (βENaC does not undergo cleavage). The bands that coprecipitated with PCSK9 correspond to the full-length forms of α- and γENaC.
      Figure thumbnail gr2
      FIGURE 2PCSK9 interacts with ENaC. A, coimmunoprecipitation of ENaC and PCSK9 in HEK 293 cells transfected with α-, β-, and γENaC (1 μg each) with or without PCSK9-V5 (3 μg). One of the ENaC subunits contained a FLAG epitope (coexpressed with the other two untagged ENaC subunits). Total cDNA was kept constant using GFP cDNA. In the top two panels, ENaC (anti-FLAG) or PCSK9 (anti-V5) was immunoprecipitated (IP) and immunoblotted (IB) as indicated. The bottom two panels show immunoblot analyses of cell lysates for ENaC and PCSK9, as indicated. Full-length and cleaved forms of PCSK9 and ENaC are indicated. The data are representative of three experiments. B, coimmunoprecipitation in HEK 293 cells transfected with a single ENaC subunit (αENaC-FLAG, βENaC-FLAG, or γENaC-FLAG, 1 μg) with or without PCSK9-V5 (1 μg). The proteins were immunoprecipitated and immunoblotted as in A. The data are representative of three experiments.
      Because α-, β-, and γENaC form a complex, we asked if each of the individual subunits could also bind to PCSK9. We cotransfected HEK 293 cells with one of the ENaC subunits, with or without PCSK9. When we immunoprecipitated each of the ENaC subunits, we detected pro-PCSK9 by immunoblot (Fig. 2B, top panel). Thus, the data indicate that PCSK9 interacts with each of the three ENaC subunits. Moreover, the interactions occur selectively between the uncleaved immature forms of PCSK9 and ENaC.

       PCSK9 Reduces ENaC Cell Surface Expression

      We asked whether PCSK9 inhibits ENaC current through a change in ENaC surface expression. In Fig. 3A, we used a biotinylation assay to detect the cell surface fraction of αENaC (coexpressed with β- and γENaC) in HEK 293 cells. Fig. 3B shows quantitative summary data. PCSK9 decreased both the full-length and proteolytically cleaved forms of αENaC at the cell surface. This decrease in surface expression corresponded to a decrease in αENaC in the total cellular pool, as detected by immunoblot analysis of cell lysates (Fig. 3A, bottom panel, and B; also see Fig. 2A). PCSK9 produced a similar decrease in expression of β- and γENaC at the cell surface and in βENaC in the total cellular pool (Fig. 3, A and B). As negative controls, PCSK9 had no effect on the abundance of heterologously expressed Nedd4-2 or endogenous β-actin (Fig. 3C). These results indicate that PCSK9 inhibits ENaC current by reducing the number of channels at the cell surface.
      Figure thumbnail gr3
      FIGURE 3PCSK9 reduces ENaC cell surface expression. A, immunoblot analyses of ENaC in the cell surface biotinylated fraction (top panel) and in the total cell lysate (bottom panel) from HEK 293 cells transfected with α-, β-, and γENaC (1 μg each, one subunit contained FLAG epitope) with or without PCSK9 (3 μg). Total cDNA was kept constant using GFP cDNA. ENaC protein in +PCSK9 group relative to -PCSK9 group is quantified by densitometry in B (mean ± S.E.; n = 3–5; *, p < 0.03). C, immunoblots of Nedd4-2-HA (anti-HA) and β-actin in HEK 293 cells transfected with Nedd4-2-HA (3 μg) with or without PCSK9 (3 μg). D, amiloride-sensitive current in Xenopus oocytes expressing human α- and γENaC with wild-type or mutant βENaC (0.2 ng each) with or without PCSK9 (0.8 ng) (mean ± S.E. relative to -PCSK9 group; n = 11–17; *, p < 0.004; n.s., p ≥ 0.05). E, immunoblot of biotinylated cell surface αY644AENaC-FLAG coexpressed with βY620A- and γY627AENaC (1 μg each) with PCSK9 or GFP (3 μg). F, immunoblot of biotinylated (top panel) and total (bottom panel) αENaC-FLAG coexpressed in HEK 293 cells with β- and γENaC (1 μg each) with GFP or PCSK9 (wild type or S386A, 3 μg). Irrelevant lanes were removed digitally.
      To determine whether PCSK9 also regulates ENaC gating, we took advantage of a mutation that locks ENaC in the open state (“DEG” mutation, βS520K) (
      • Snyder P.M.
      • Bucher D.B.
      • Olson D.R.
      Gating induces a conformational change in the outer vestibule of ENaC.
      ). If PCSK9 inhibits ENaC in part through a change in gating, this mutation should blunt the effect. However, we found that PCSK9 inhibited mutant ENaC to the same extent as wild-type ENaC (Fig. 3D). This finding indicates that the changes we observed in ENaC surface expression are sufficient to explain PCSK9 inhibition of ENaC.
      Prior work has shown that the PY motifs located in the C termini of ENaC subunits play an important role in trafficking (
      • Snyder P.M.
      Down-regulating destruction: phosphorylation regulates the E3 ubiquitin ligase Nedd4-2.
      ). The PY motifs function as binding sites for Nedd4-2, an E3 ubiquitin ligase that catalyzes ENaC ubiquitination. This functions as a signal to induce ENaC endocytosis and degradation in lysosomes. Importantly, mutations in the PY motifs cause Liddle's syndrome, an inherited form of hypertension. To test whether the PY motifs are required for ENaC regulation by PCSK9, we mutated the conserved tyrosine residue within the motif of each ENaC subunit (αY644A, βY620A, and γY627A). In Fig. 3E, we found that PCSK9 reduced surface expression of the mutant ENaC. This finding suggests that PCSK9 regulates ENaC surface expression through a pathway that is independent of the PY motifs and Nedd4-2.
      To test whether protease activity is needed for PCSK9 to reduce ENaC surface expression, we introduced a mutation that abolishes proteolytic activity (S386A) (
      • McNutt M.C.
      • Lagace T.A.
      • Horton J.D.
      Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells.
      ). PCSK9S386A decreased αENaC expressed at the cell surface and in the total cellular pool similar to wild-type PCSK9 (Fig. 3F). Thus, the catalytic activity is not required for PCSK9 to regulate ENaC, similar to its regulation of the LDLR.

       PCSK9 Increases ENaC Degradation

      To further investigate the mechanism by which PCSK9 reduced ENaC cell surface expression, we asked whether PCSK9 alters ENaC degradation using a cycloheximide chase assay. HEK 293 cells expressing ENaC and PCSK9 or GFP (control) were treated with cycloheximide for 0–120 min to inhibit protein synthesis. In Fig. 4, A and B, we detected and quantitated the remaining αENaC-FLAG at each time point by immunoblot analysis. In the absence of PCSK9, there was no significant decrease in αENaC over the 120-min time course of the experiment. In contrast, in cells transfected with PCSK9, there was a time-dependent decrease in αENaC. This finding indicates that PCSK9 accelerates the rate of ENaC degradation.
      Figure thumbnail gr4
      FIGURE 4PCSK9 increases ENaC degradation. A, immunoblot analyses of αENaC-FLAG in HEK 293 cells cotransfected with β- and γENaC (1 μg each) with or without PCSK9 (3 μg). The cell were treated with cycloheximide (CHX) (10 μg/ml) for 0–120 min prior to lysis. B, quantification αENaC, relative to 0 min time point (mean ± S.E.; n = 6; *, p < 0.007). C, immunoblot analyses of αENaC-FLAG in HEK 293 cells cotransfected with β- and γENaC (1 μg each) with or without PCSK9 (3 μg). The cells were treated with N-acetyl-Leu-Leu-norleucinal (ALLN) (10 μm), NH4Cl (5 mm), or vehicle for 2 h prior to lysis. Data are representative of at least three experiments.
      To localize the site of the PCSK9-induced ENaC degradation, we incubated cells with inhibitors of the proteasome (N-acetyl-Leu-Leu-norleucinal) or lysosomes (NH4Cl). We found that N-acetyl-Leu-Leu-norleucinal partially reversed the effect of PCSK9 on αENaC expression, whereas NH4Cl had no effect (Fig. 4C). Together, the data indicate that PCSK9 reduces ENaC surface expression in part by enhancing its degradation in the proteasome.

       Effect of PCSK9 on ENaC Exocytosis

      ENaC surface expression is controlled through a balance between exocytosis of newly formed channels, endocytosis of cell surface channels, and recycling of channels in the endocytic pathway. Because PCSK9 increased ENaC degradation, it seemed likely that PCSK9 would reduce the pool of ENaC available for exocytosis. To test this possibility, we used a functional strategy we reported previously (
      • Snyder P.M.
      Liddle's syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na(+) channel to the cell surface.
      ). We covalently modified the cell surface pool of ENaC and then measured the rate of appearance of unmodified channels at the cell surface. For these experiments, we placed a cysteine in the pore of γENaC (G536C) (
      • Snyder P.M.
      • Olson D.R.
      • Bucher D.B.
      A pore segment in DEG/ENaC Na(+) channels.
      ,
      • Snyder P.M.
      Liddle's syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na(+) channel to the cell surface.
      ). When the mutant γ subunit was coexpressed in FRT epithelia with wild-type α- and βENaC, MTSET irreversibly blocked the channel by modifying the introduced cysteine, as shown in the representative current traces in Fig. 5, A and B. Because MTSET is not membrane-permeable, the intracellular ENaC pool was protected from modification. Following removal of MTSET from the bathing solution, we measured the increase in ENaC current over time as an assay of exocytosis of unmodified/unblocked channels. Fig. 5C shows the averaged time courses of current recovery, and the time constants are shown in D. PCSK9 reduced the maximal increase in current recovery over time but did not significantly alter the time course of the increase, as reflected by the lack of difference in the rate constant. These results are consistent with a reduction in the size of the ENaC pool available for exocytosis. However, the observed decrease in current recovery could also be explained by an increase in the rate of ENaC endocytosis. We therefore tested the effect of PCSK9 on ENaC endocytosis.
      Figure thumbnail gr5
      FIGURE 5PCSK9 decreases ENaC exocytosis. Short-circuit currents were recorded in FRT epithelia transfected with α-, β-, and γG536CENaC subunits (0.16 μg each) with or without PCSK9 (0.5 μg). A and B, representative current traces. MTSET (1 mm) was added to the apical membrane as indicated by the black bars. C, summary data for the amiloride-sensitive current in the absence and presence of PCSK9 (mean ± S.E., n = 7). D, time constants for single exponential fit of the data in C (mean ± S.E.; n = 7; n.s., p ≥ 0.05).

       PCSK9 Does Not Alter Trafficking of the Cell Surface ENaC Pool

      In Fig. 6, we quantitated endocytosis of mature proteolytically cleaved ENaC using a method that we described previously (
      • Kabra R.
      • Knight K.K.
      • Zhou R.
      • Snyder P.M.
      Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels.
      ). We mutated the two furin consensus sites in the extracellular domain of αENaC to prevent cleavage by furin (αCl-2) and then treated the cells briefly with trypsin to generate a pool of proteolytically cleaved channels (65-kDa band). After removal of trypsin, we incubated the cells at 37 °C for 0–60 min to allow endocytosis of the cleaved channels and then biotinylated and detected channels remaining at the cell surface. The disappearance of the 65-kDa band reflects the rate of ENaC endocytosis. Because newly synthesized mutant channels reaching the cell surface are uncleaved, they can be distinguished from the cleaved channels undergoing endocytosis. As shown in Fig. 6, A and B, the 65-kDa cleaved band was rapidly removed from the cell surface with a half-life of ∼15 min, consistent with our prior work (
      • Kabra R.
      • Knight K.K.
      • Zhou R.
      • Snyder P.M.
      Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels.
      ). However, PCSK9 did not alter the rate of ENaC disappearance from the cell surface. Thus, PCSK9 does not alter ENaC surface expression through a change in endocytosis.
      Figure thumbnail gr6
      FIGURE 6PCSK9 does not alter ENaC endocytosis. A, immunoblot analysis (anti-FLAG) of biotinylated αCl-2 ENaC-FLAG in HEK 293 cells cotransfected with β- and γENaC (1 μg each) with or without PCSK9 (3 μg). The cells were treated with 5 μg/ml trypsin for 5 min, incubated at 37 °C for 0–60 min, and then biotinylated. B, quantification of the cleaved αENaC band relative to 0 min (mean ± S.E., n = 5).
      Following endocytosis, ENaC can either recycle back to the cell surface or traffic to lysosomes for degradation. To test whether PCSK9 alters ENaC surface expression in part by regulating this sorting step, we measured the effect of PCSK9 on degradation of the cell surface pool of ENaC. In HEK 293 cells expressing ENaC, we pulse-labeled the cell surface fraction of channels with biotin, incubated the cells at 37 °C for 0–120 min, and then quantitated the remaining (non-degraded) biotinylated channels. PCSK9 did not increase the rate of degradation of biotinylated αENaC (Fig. 7, A and B). Rather, it slightly delayed degradation, which may partially counter the effect of PCSK9 on ENaC degradation in the biosynthetic pathway.
      Figure thumbnail gr7
      FIGURE 7PCSK9 does not alter degradation of cell surface ENaC. A, immunoblots (anti-FLAG) of biotinylated αENaC in HEK 293 cells transfected with αENaC-FLAG, βENaC, and γENaC (1 μg each) with or without PCSK9 (3 μg). Cell surface proteins were pulse-labeled with biotin and then the cells were incubated at 37 °C for 0–120 min. B, biotinylated αENaC at each time was quantified relative to 0 min (mean ± S.E.; n = 3; *, p < 0.05).

      DISCUSSION

      Recent work has focused on the regulation of epithelial Na+ transport by proteases, including furin, a member of the proprotein convertase family (
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
      ). Here we found that ENaC is regulated by PCSK9, another member of this protease family. However, furin and PCSK9 have opposite effects on ENaC current and they regulate ENaC through different mechanisms. In contrast to furin, which activates ENaC by proteolytic cleavage of the extracellular domains of the α and γ subunits, PCSK9 inhibits ENaC by reducing its cell surface expression. Moreover, unlike furin, PCSK9 regulates ENaC independent of its protease activity.
      The data indicate that PCSK9 reduces ENaC surface expression primarily by increasing its degradation in the biosynthetic pathway, which reduces the pool of ENaC available for exocytosis. Consistent with this concept, we found that PCSK9 decreased ENaC exocytosis and increased the rate of ENaC degradation in the proteasome. Moreover, PCSK9 had no effect on the rate of ENaC endocytosis or its degradation in the endocytic pathway. Coimmunoprecipitation studies suggest that ENaC and PCSK9 interact with one another in their immature (uncleaved) states, likely prior to ENaC cleavage in the Golgi apparatus (although we cannot exclude the possibility that proteolytic cleavage induces conformation changes that prohibit binding). Together, these findings are most consistent with a model in which PCSK9 enhances endoplasmic reticulum associated degradation of ENaC.
      The mechanism by which PCSK9 regulates ENaC shares some similarities to its regulation of the LDLR. In both cases, PCSK9 reduces surface expression through a change in trafficking, culminating in increased degradation. Likewise, both occur independent of PCSK9 protease activity. However, there are also important differences. Contrary to regulation of ENaC in the biosynthetic pathway, PCSK9 predominately regulates the LDLR in the endocytic pathway (
      • Nassoury N.
      • Blasiole D.A.
      • Tebon Oler A.
      • Benjannet S.
      • Hamelin J.
      • Poupon V.
      • McPherson P.S.
      • Attie A.D.
      • Prat A.
      • Seidah N.G.
      The cellular trafficking of the secretory proprotein convertase PCSK9 and its dependence on the LDLR.
      ,
      • Zhang D.W.
      • Lagace T.A.
      • Garuti R.
      • Zhao Z.
      • McDonald M.
      • Horton J.D.
      • Cohen J.C.
      • Hobbs H.H.
      Binding of proprotein convertase subtilisin/kexin type 9 to epidermal growth factor-like repeat A of low density lipoprotein receptor decreases receptor recycling and increases degradation.
      ). This suggests that PCSK9 can function in multiple cellular compartments. There are also differences in the mechanisms of binding. PCSK9 interacts with the LDLR through two interfaces. Crystallization revealed that the catalytic domain of PCSK9 binds to the epidermal growth factor-like A domain in the LDLR (
      • Kwon H.J.
      • Lagace T.A.
      • McNutt M.C.
      • Horton J.D.
      • Deisenhofer J.
      Molecular basis for LDL receptor recognition by PCSK9.
      ). A recent report found a second interaction between the PCSK9 C-terminal domain and the LDLR ligand binding domain, which was required for regulation (
      • Yamamoto T.
      • Lu C.
      • Ryan R.O.
      A two-step binding model of PCSK9 interaction with the low density lipoprotein receptor.
      ). Importantly, ENaC lacks homologous motifs, indicating that it interacts with PCSK9 through a novel binding mechanism. This interaction could be direct or could occur indirectly through an adaptor protein.
      The proprotein convertase furin regulates ENaC by proteolytic cleavage of the extracellular domains of α- and γENaC, which releases inhibitory peptides (
      • Kleyman T.R.
      • Carattino M.D.
      • Hughey R.P.
      ENaC at the cutting edge: regulation of epithelial sodium channels by proteases.
      ). In this manner, furin regulates ENaC gating, converting near-silent channels into active channels. However, there may be an additional level of complexity. Furin also proteolytically cleaves PCSK9, which inactivates it (
      • Essalmani R.
      • Susan-Resiga D.
      • Chamberland A.
      • Abifadel M.
      • Creemers J.W.
      • Boileau C.
      • Seidah N.G.
      • Prat A.
      In vivo evidence that furin from hepatocytes inactivates PCSK9.
      ). Thus, through a decrease in PCSK9 activity, furin could increase ENaC cell surface expression. This raises the interesting possibility that furin regulates ENaC through dual effects on channel trafficking and gating.
      Defects in ENaC regulation are responsible for the majority of the known genetic forms of hypertension, which is an important risk factor for coronary heart disease and other cardiovascular diseases. Thus, PCSK9 could modulate cardiovascular risk in part through its regulation of ENaC. We speculate that a decrease in PCSK9 activity would increase renal Na+ absorption and, therefore, raise the risk of hypertension and associated cardiovascular disease. However, such a mechanism would counter the previously reported effect of PCSK9 mutations on cardiovascular risk. Activating mutations were found to increase the risk, whereas loss-of-function mutations reduced the risk (
      • Horton J.D.
      • Cohen J.C.
      • Hobbs H.H.
      Molecular biology of PCSK9. Its role in LDL metabolism.
      ,
      • Seidah N.G.
      • Prat A.
      The proprotein convertases are potential targets in the treatment of dyslipidemia.
      ,
      • Maxwell K.N.
      • Breslow J.L.
      Proprotein convertase subtilisin kexin 9. The third locus implicated in autosomal dominant hypercholesterolemia.
      ,
      • Cohen J.C.
      • Boerwinkle E.
      • Mosley Jr., T.H.
      • Hobbs H.H.
      Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.
      ,
      • McNutt M.C.
      • Lagace T.A.
      • Horton J.D.
      Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells.
      ). These effects are thought to occur through changes in expression of the LDLR, which produce changes in serum levels of cholesterol. Thus, it is possible that PCSK9 regulation of ENaC and the LDLR have opposing effects on cardiovascular risk. On the other hand, our data suggest that PCSK9 regulates ENaC and the LDLR through different binding sites and different mechanisms. Thus, the PCSK9 mutations that disrupt LDLR regulation may have dissimilar effects on ENaC. Additional work will be required to test whether naturally occurring mutations in PCSK9 alter ENaC trafficking, renal Na+ homeostasis, and blood pressure.

      Acknowledgments

      We thank Danielle Wentzlaff, Caitlin Digman, Zeru Peterson, Abigail Hamilton, and Nicole Pearson for assistance. We also thank the University of Iowa DNA Core Facility for reagents and DNA sequencing.

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