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

Received for publication, March 19, 2012, and in revised form, April 5, 2012 Published, JBC Papers in Press, April 9, 2012, DOI 10.1074/jbc.M112.363382

Vikas Sharotri, Daniel M. Collier, Diane R. Olson, Ruifeng Zhou, and Peter M. Snyder1

From the Departments of Internal Medicine and Molecular Physiology and Biophysics, University of Iowa Carver College ofMedicine, Iowa City, Iowa 52242

Background: The epithelial Na� channel ENaC functions as a pathway for Na� absorption across epithelia.Results: PCSK9 reduced ENaC expression at the cell surface by enhancing its proteasomal degradation.Conclusion: PCSK9 inhibits ENaC-mediated Na� absorption.Significance: These findings provide new insights into mechanisms that regulate Na� homeostasis and blood pressure.

The epithelial Na� channel (ENaC) is critical for Na� ho-meostasis and blood pressure control. Defects in its regulationcause inherited forms of hypertension and hypotension. Previ-ous work found that ENaC gating is regulated by proteasesthrough cleavage of the extracellular domains of the � and �subunits. Here we tested the hypothesis that ENaC is regulatedby proprotein convertase subtilisin/kexin type 9 (PCSK9), a pro-tease that modulates the risk of cardiovascular disease. PCSK9reduced ENaC current in Xenopus oocytes and in epithelia.This occurred through a decrease in ENaC protein at the cellsurface and in the total cellular pool, an effect that did notrequire the catalytic activity of PCSK9. PCSK9 interactedwith all three ENaC subunits and decreased their traffickingto the cell surface by increasing proteasomal degradation. Incontrast to its previously reported effects on the LDL recep-tor, PCSK9 did not alter ENaC endocytosis or degradation ofthe pool of ENaC at the cell surface. These results support arole for PCSK9 in the regulation of ENaC trafficking in thebiosynthetic pathway, likely by increasing endoplasmic retic-ulum-associated degradation. By reducing ENaC channelnumber, PCSK9 could modulate epithelial Na� absorption, amajor contributor to blood pressure control.

The epithelial Na� channel (ENaC)2 plays an important rolein absorption of Na� across epithelia, including the kidney,collecting duct and connecting tubule, lung, distal colon, andsweat duct (reviewed in Refs. 1, 2). A heterotrimer composed ofthree homologous subunits (�, �, and �), ENaC is expressed atthe apicalmembranewhere it forms a pathway forNa� to enterthe cell. Na� leaves the cell at the basolateral membrane via theNa�-K�-ATPase, which completes the pathway for Na�

absorption. This process is critical to control extracellular vol-ume and tomaintain the composition and quantity of epithelial

surface liquid. This is illustrated by several diseases. For exam-ple, ENaCmutations that slow its retrieval from the cell surfacecause an inherited form of hypertension (Liddle’s syndrome),resulting from excessive renal Na� absorption (3–5). Defects inENaC regulation are responsible for most of the known geneticforms of hypertension (6). Conversely, loss of function muta-tions cause pseudohypoaldosteronism type I, a disorder of renalNa� wasting (7). In the lung, defects in ENaC activity causepulmonary edema and may contribute to the pathogenesis ofcystic fibrosis (8).Previouswork indicates that ENaC is regulated by serine pro-

teases (reviewed in Ref. 9). Furin, a member of the proproteinconvertase family, cleaves the extracellular domain of�ENaCatbasic motifs, removing a 26-amino acid fragment (10). In�ENaC, furin cleaves the extracellular domain at a single siteand a second, more distal site is cleaved by additional proteases(e.g.CAP1/prostasin, plasmin, elastase), releasing a fragment of�43 amino acids (11–15). Proteolytic cleavage of �- and�ENaC converts quiescent channels into active Na�-conduct-ing channels. This activation occurs by relieving the channelfrom inhibition by extracellular Na� (“Na� self-inhibition”)(16). Proteolytic cleavage of ENaC is a regulated process. Forexample, cleavage is inhibited by increased intracellular Na�,providing a negative feedback mechanism to regulate Na�

absorption (17). Conversely, cleavage is enhanced by Na�

depletion and aldosterone infusion (18, 19). Cleavage is alsodisrupted in pathological states. In Liddle’s syndrome, cleavageis increased, likely through prolonged exposure of ENaC to pro-teases present at the cell surface (5). There is also evidence tosuggest that ENaC cleavage is increased in nephrotic syndrome(15, 20) and cystic fibrosis (21, 22).Because proteolytic cleavage modulates ENaC gating, there

has been considerable interest in identifying additional pro-teases that regulate ENaC. The proprotein convertase familyhas nine members, including furin (23). In this work, we inves-tigated a potential role for another member of this family, pro-protein convertase subtilisin/kexin type 9 (PCSK9) (24). Con-sistent with a potential role in ENaC regulation, PCSK9 isexpressed in the kidney and lung (24). It is synthesized as a72-kDa immature precursor that undergoes autocatalyticcleavage in the endoplasmic reticulum to generate a 63-kDamature protein (25). The cleavedN-terminal fragment remains

* This work was supported, in whole or in part, by National Institutes of HealthGrant HL058812 (to P. M. S.). This work was also supported by fellowshipgrants from the American Heart Association (to V. S. and D. M. C.).

1 To whom correspondence should be addressed: 371 EMRB, University ofIowa, Iowa City, IA 52242. E-mail: peter-snyder@uiowa.edu.

2 The abbreviations used are: ENaC, epithelial Na� channel; PCSK9, propro-tein convertase subtilisin/kexin type 9; LDLR, LDL receptor; FRT, Fischerrat thyroid; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonatebromide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 23, pp. 19266 –19274, June 1, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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associated with the mature protein and is necessary for itssecretion, allowing it to circulate in the blood (26).Previous work has focused on the role of PCSK9 in the regu-

lation of the LDL receptor (LDLR). By reducing expression ofthe LDLR at the cell surface, PCSK9 increases serum levels ofLDL cholesterol (25, 27, 28). Rare gain-of-function PCSK9mutations cause hypercholesterolemia and increase the risk ofcoronary heart disease, whereas loss-of-function mutationscause hypocholesterolemia and protect against heart disease(29–33). The mechanisms by which PCSK9 alters LDLR sur-face expression are not completely understood. SecretedPCSK9 (or recombinant PCSK9 added to the extracellularmedium) binds to the LDLR and undergoes endocytosis (26, 27,34–36). In the endocytic pathway, PCSK9 increases lysosomaldegradation of the LDLR. Although secreted PCSK9 regulatesLDLR trafficking, additional evidence suggests that PCSK9mayalso induce LDLR degradation through an intracellular route(37). Interestingly, although PCSK9 induces degradation of theLDLR, its protease activity is not required (33, 38). Thus, it hasbeen proposed that PCSK9 regulates the LDLR through a chap-erone mechanism, rather than through its function as a prote-ase. Although it seems clear that the PCSK9 regulates the LDLRand two closely related receptors (very low density lipoproteinreceptor and apolipoprotein E receptor 2 (38)), additional sub-strates for PCSK9 have not been identified. Here we show thatPCSK9 regulates ENaC and we explore the mechanisms thatunderlie this regulation.

EXPERIMENTAL PROCEDURES

DNAConstructs—Human�ENaC,�ENaC, and �ENaCwerecloned in pMT3 as described previously (39, 40). Mutationswere generated by site-directed mutagenesis (QuikChange;Stratagene). �ENaC-FLAG, �ENaC-FLAG, and �ENaC-FLAGwere generated by insertion of a FLAG epitope (DYKDDDDK)at theC terminus (5, 41). HumanPCSK9-V5was a generous giftfrom Nabil Seidah (24), and Nedd4-2-HA was generated asdescribed (42). Mutations were generated (QuikChange, Strat-agene) in �ENaC (Y644A, R175A, R177A, R178A, R181A,R190A, R192A, R201A, R204A), �ENaC (Y620A), and �ENaC(Y627A) (G536C) as described previously (3, 43, 44) and inPCSK9 (S386A). All cDNAs were sequenced in the Universityof Iowa DNA Core Facility.Electrophysiology in Xenopus Oocytes—Oocytes were har-

vested from Xenopus laevis females. They were treated for 1 hwith 0.75 mg/ml type IV collagenase (Sigma) in Ca2�-freeND-96 (96mMNaCl, 2mMKCl, 1mMMgCl2, 5mMHEPES (pH7.4)) andmanually defolliculated (45–47). The cell nucleus wasinjected with cDNAs encoding either human �-, �- (wild-typeor S520K), and �ENaC (0.2 ng each) or with human ASIC1 (0.6ng) along with PCSK9 (0–2 ng). Total injected cDNA was heldconstant using pMT3-SEAP (48). Following injection, oocyteswere incubated at 18 °C inmodified Barth’s saline (88mMNaCl,1mMKCl, 0.33mMCa(NO3)2, 0.41mMCaCl2, 0.82mMMgSO4,2.4 mM NaHCO3, 10 mM HEPES, 50 �g/ml gentamicin sulfate,10 �g/ml sodium penicillin, 10 �g/ml streptomycin sulfate (pH7.4)) 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 Pow-erlab interface (ADInstruments), and recorded and analyzedwith Chart software (ADInstruments). The cells were bathed in116mMNaCl, 2mMKCl, 0.4mMCaCl2, 1mMMgCl2, and 5mM

HEPES (pH 7.4 or 5). The amiloride-sensitive ENaC currentwas measured by adding 10 �M amiloride to the bathing solu-tion. ASIC1 currents were detected by addition of pH 5 to thebathing solution.Electrophysiology in Epithelia—To test the effect of PCSK9

on ENaC current in epithelia, Fischer rat thyroid (FRT) cellswere cultured on permeable filter supports (Millicell PCF,0.4-�m pore size, 12-mm diameter) in F-12 Coon’s medium(Sigma) with 5% fetal calf serum (Sigma), 100 units/ml penicil-lin, and 100�g/ml streptomycin at 37 °C.Cellswere transfectedwith �-, �-, and �ENaC (0.03 �g each) with or without PCSK9(0–0.9 �g) using TFX50 (Promega) as described previously(44, 49). Total cDNA was kept constant using GFP cDNA(which does not alter ENaC current). Two days after trans-fection, the current was measured in Ussing chambersunder short-circuit conditions using an EC-825 amplifier(Warner Instruments), digitized with a Powerlab interface(ADInstruments), and recorded and analyzed with Chartsoftware (ADInstruments). The apical and basolateral sur-faces 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 �M) was added to the apicalsolution to quantitate ENaC current.For exocytosis experiments, FRT cells were transfected with

�ENaC, �ENaC, and �536CENaC (0.167 �g each subunit) withPCSK9 or GFP cDNA (0.5 �g) (43, 49). Channels at the cellsurface were irreversibly blocked by covalent modification ofthe introduced cysteine with 1 mM [2-(trimethylammonium)-ethyl]methanethiosulfonate bromide (MTSET). Followingremoval ofMTSET,wemeasured the rate of current increase toquantitate exocytosis of unblocked channels. Time constants(�) were determined by fitting the data to single-exponentialequations using IGOR Pro 6.01 software.Coimmunoprecipitation—HEK 293T cells were cultured in

Dulbecco’s modified Eagle’s medium. To test for interactionsbetween ENaC and PCSK9, the cells were transfected with�ENaC, �ENaC, and �ENaC (individually or together, 1 �geach) and PCSK9-V5 or GFP (1 or 3 �g) using Lipo-fectamine2000 (Invitrogen) (5, 41). One ENaC subunit con-tained a FLAG epitope. Two days after transfection, the cellswere lysed in Nonidet P-40 lysis buffer (0.4% sodium deoxy-cholate, 1% Nonidet P-40, 63 mM EDTA, 50 mM Tris-HCl (pH8), and protease inhibitor mixture (Sigma)). 500 �g of cellularprotein was immunoprecipitated with anti-FLAG M2 affinitygel (Sigma) or anti-V5 antibody (Invitrogen) with immobilizedprotein A (Pierce) beads overnight at 4 °C. Following SDS-PAGE, ENaC and PCSK9 were detected by immunoblot analy-sis using anti-FLAGM2monoclonal antibody-peroxidase con-jugate (1:5000, Sigma) at 1:5000 dilution or anti-V5 antibody(1:5000) and enhanced chemiluminescence (ECL Plus, GEHealthcare).Biotinylation—To quantitate ENaC expression at the cell

surface, HEK 293 cells expressing �-, �-, and �ENaC (FLAGepitope on one subunit) and PCSK9 or GFP were washed with

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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 (5). Excess biotin wasquenched 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 thencentrifuged at 14,000 rpm for 10min to remove insolublemate-rial. Biotinylated proteins were isolated with NeutrAvidin-aga-rose beads (Pierce) overnight at 4 °C. Following extensivewash-ing, 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. BiotinylatedENaC and ENaC in the total cellular lysate were detected byimmunoblot using anti-FLAGM2-peroxidase-conjugated anti-body (1:5000, Sigma) and enhanced chemiluminescence (ECLPlus, GEHealthcare) and quantitated by densitometry (ImageJ)using non-saturated exposures.Degradation—To measure the rate of ENaC degradation,

HEK 293 cells transfected with �ENaC-FLAG, �ENaC, and�ENaCwith PCSK9orGFPwere incubatedwith cycloheximide(10 �g/ml) for 0–120 min. Remaining �ENaC-FLAG at eachtime point was detected by immunoblot (anti-FLAG M2-per-oxidase-conjugated antibody) and quantitated by densitome-try. To identify the location of degradation, cells were treatedwith 10 �M N-acetyl-Leu-Leu-norleucinal or 5 mM NH4Cl.To measure the rate of degradation of the cell surface frac-

tion of ENaC, HEK 293 cells transfected with �ENaC-FLAG,�ENaC, and �ENaC with PCSK9 or GFP were biotinylated onice and then incubated at 37 °C for 0–120 min (5, 41). Biotiny-lated �ENaC-FLAG was isolated using NeutrAvidin-agarose,detected by immunoblot (anti-FLAG M2-peroxidase-conju-gated antibody), and quantitated by densitometry.Endocytosis—To measure the rate of ENaC endocytosis, we

used a previously described �ENaC construct (�Cl-2) in whichmultiple arginines were simultaneously mutated to preventproteolytic cleavage by furin but to retain the ability to becleaved by trypsin (R175A, R177A, R178A, R181A, R190A,R192A, R201A, and R204A) (44). HEK 293 cells were trans-fected with �Cl-2 ENaC-FLAG, �ENaC, and �ENaC withPCSK9 or GFP were incubated with trypsin (5 �g/ml) for 5minat 37 °C to generate a pool of cleaved channels at the cell surface(9)(44). The cells werewashed three timeswith cold PBS-CMtoremove trypsin, incubated at 37 °C for 0–60min to allow endo-cytosis of cleaved channels, and then placed on ice. Cleavedchannels remaining at the cell surface were labeled with biotin,isolated with NeutrAvidin-agarose, detected by immunoblotanalysis (anti-FLAGM2-peroxidase-conjugated antibody), andquantitated by densitometry.

RESULTS

PCSK9 Inhibits ENaC—We tested the effect of PCSK9 onENaC current utilizing two expression systems. First, weinjected Xenopus oocytes with �-, �-, and �ENaC cDNA togenerate amiloride-sensitive Na� currents (Fig. 1A). We foundthat coexpression of PCSK9 decreased the Na� current in adose-dependent manner (Fig. 1, A and B).As a second strategy, we tested the effect of PCSK9 on the

ENaC current in epithelia. Transfection of FRT epithelia with

�-, �-, and �ENaC resulted in amiloride-sensitive short-circuitcurrents (Fig. 1C). Cotransfection with PCSK9 produced adose-dependent decrease in ENaC current (Fig. 1, C and D),similar to our results in oocytes. Thus, PCSK9 inhibited ENaCin two independent experimental systems.We also tested the effect of PCSK9 on a related DEG/ENaC

channel, ASIC1. PCSK9 reduced the proton-activated ASIC1current by 24% inXenopus oocytes (Fig. 1, E and F), less than itseffect on ENaC.PCSK9 Interacts with ENaC—To begin to investigate the

mechanism by which PCSK9 inhibits ENaC current, we testedwhether PCSK9 and ENaC interact with one another. In Fig.2A, we transfected HEK 293 cells with �-, �-, and �ENaC(one of the subunits contained a FLAG epitope) along withPCSK9 (V5 epitope) and examined protein interactions using acoimmunoprecipitation assay. When we immunoprecipitated�ENaC, we detected coprecipitated PCSK9 in cells cotrans-fected with ENaC and PCSK9 but not in cells transfected indi-vidually with either ENaC or PCSK9 (Fig. 2A, first panel). Like-wise, we detected PCSK9 when we immunoprecipitated �- or

FIGURE 1. PCSK9 inhibits ENaC current. A and B, Xenopus oocytes werenuclear-injected with cDNAs encoding human �-, �-, and �ENaC (0.2 ng each)and PCSK9 (0 –2 ng). A, representative current traces (0.2 ng PCSK9). 10 �M

amiloride (Amil) was added to the bathing solution as indicated by the blackbar. B, summary plot of amiloride-sensitive current versus amount of injectedPCSK9 cDNA (mean � S.E. relative to 0 PCSK9 group, n � 5–26). C and D, FRTepithelia were transfected with �-, �-, and �ENaC (0.03 �g each) and PCSK9(0 – 0.9 �g). C, representative short-circuit current traces. 10 �M amiloride wasadded to the apical bathing solution as indicated by the black bar. D, sum-mary plot of amiloride-sensitive current versus the amount of transfectedPCSK9 (mean � S.E. relative to 0 PCSK9 group, n � 9 –12). E and F, Xenopusoocytes were nuclear-injected with cDNAs encoding human ASIC1 (0.6 ng)and PCSK9 or control plasmid (0.8 ng). E, representative current traces. Thebath was perfused with pH 5 solution as indicated by the black bar. F, sum-mary plot of proton-activated current (mean � S.E.; n � 9; *, p � 0.03).

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�ENaC (Fig. 2A, first panel). There are two forms of PCSK9,full-length pro-PCSK9 (72 kDa) and autocatalytically cleavedPCSK9 (63 kDa) (Fig. 2A, third panel) (25). ENaC selectivelycoprecipitated the pro-PCSK9 form (Fig. 2A, first panel). Usinga reciprocal strategy we found that �-, �-, and �ENaC eachcoprecipitated when we immunoprecipitated PCSK9 (Fig. 2A,second panel). In the immunoblot analysis, we observed twobands for �- and �ENaC, which correspond to the full-length(immature) and proteolytically cleaved (mature) forms, respec-tively (�ENaC does not undergo cleavage). The bands thatcoprecipitated with PCSK9 correspond to the full-length formsof �- and �ENaC.Because �-, �-, and �ENaC form a complex, we asked if each

of the individual subunits could also bind to PCSK9. Wecotransfected HEK 293 cells with one of the ENaC subunits,with or without PCSK9.When we immunoprecipitated each ofthe ENaC subunits, we detected pro-PCSK9 by immunoblot(Fig. 2B, top panel). Thus, the data indicate that PCSK9 inter-acts with each of the three ENaC subunits.Moreover, the inter-

actions occur selectively between the uncleaved immatureforms of PCSK9 and ENaC.PCSK9 Reduces ENaC Cell Surface Expression—We asked

whether PCSK9 inhibits ENaC current through a change inENaC surface expression. In Fig. 3A, we used a biotinylationassay to detect the cell surface fraction of �ENaC (coexpressedwith �- and �ENaC) in HEK 293 cells. Fig. 3B shows quantita-tive summary data. PCSK9 decreased both the full-length andproteolytically cleaved forms of �ENaC at the cell surface. Thisdecrease in surface expression corresponded to a decrease in�ENaC in the total cellular pool, as detected by immunoblotanalysis of cell lysates (Fig. 3A, bottom panel, andB; also see Fig.2A). PCSK9 produced a similar decrease in expression of�- and�ENaC at the cell surface and in �ENaC in the total cellularpool (Fig. 3,A andB). As negative controls, PCSK9had no effecton the abundance of heterologously expressed Nedd4-2 orendogenous �-actin (Fig. 3C). These results indicate thatPCSK9 inhibits ENaC current by reducing the number of chan-nels at the cell surface.To determine whether PCSK9 also regulates ENaC gating,

we took advantage of a mutation that locks ENaC in the openstate (“DEG”mutation, �S520K) (50). If PCSK9 inhibits ENaC inpart through a change in gating, this mutation should blunt theeffect. However, we found that PCSK9 inhibited mutant ENaCto the same extent as wild-type ENaC (Fig. 3D). This findingindicates that the changes we observed in ENaC surface expres-sion 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(51). The PYmotifs function as binding sites forNedd4-2, an E3ubiquitin ligase that catalyzes ENaC ubiquitination. This func-tions as a signal to induce ENaC endocytosis and degradation inlysosomes. Importantly, mutations in the PY motifs cause Lid-dle’s syndrome, an inherited form of hypertension. To testwhether the PY motifs are required for ENaC regulation byPCSK9, we mutated the conserved tyrosine residue within themotif of eachENaC subunit (�Y644A,�Y620A, and�Y627A). In Fig.3E, we found that PCSK9 reduced surface expression of themutant ENaC. This finding suggests that PCSK9 regulatesENaC surface expression through a pathway that is indepen-dent of the PY motifs and Nedd4-2.To test whether protease activity is needed for PCSK9 to

reduce ENaC surface expression, we introduced a mutationthat abolishes proteolytic activity (S386A) (33). PCSK9S386Adecreased �ENaC expressed at the cell surface and in the totalcellular pool similar to wild-type PCSK9 (Fig. 3F). Thus, thecatalytic 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 surfaceexpression, we asked whether PCSK9 alters ENaC degradationusing a cycloheximide chase assay. HEK 293 cells expressingENaC and PCSK9 orGFP (control) were treated with cyclohex-imide for 0–120 min to inhibit protein synthesis. In Fig. 4, Aand B, we detected and quantitated the remaining �ENaC-FLAG at each time point by immunoblot analysis. In theabsence of PCSK9, there was no significant decrease in �ENaCover the 120-min time course of the experiment. In contrast, in

FIGURE 2. PCSK9 interacts with ENaC. A, coimmunoprecipitation of ENaCand PCSK9 in HEK 293 cells transfected with �-, �-, and �ENaC (1 �g each) withor without PCSK9-V5 (3 �g). One of the ENaC subunits contained a FLAGepitope (coexpressed with the other two untagged ENaC subunits). TotalcDNA 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 celllysates for ENaC and PCSK9, as indicated. Full-length and cleaved forms ofPCSK9 and ENaC are indicated. The data are representative of three experi-ments. B, coimmunoprecipitation in HEK 293 cells transfected with a singleENaC subunit (�ENaC-FLAG, �ENaC-FLAG, or �ENaC-FLAG, 1 �g) with or with-out PCSK9-V5 (1 �g). The proteins were immunoprecipitated and immuno-blotted as in A. The data are representative of three experiments.

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cells transfected with PCSK9, there was a time-dependentdecrease in �ENaC. This finding indicates that PCSK9 acceler-ates the rate of ENaC degradation.To localize the site of the PCSK9-inducedENaCdegradation,

we incubated cells with inhibitors of the proteasome (N-acetyl-Leu-Leu-norleucinal) or lysosomes (NH4Cl). We found thatN-acetyl-Leu-Leu-norleucinal partially reversed the effect ofPCSK9 on �ENaC expression, whereas NH4Cl had no effect(Fig. 4C). Together, the data indicate that PCSK9 reduces ENaCsurface expression in part by enhancing its degradation in theproteasome.Effect of PCSK9 on ENaC Exocytosis—ENaC surface expres-

sion is controlled through a balance between exocytosis ofnewly formed channels, endocytosis of cell surface channels,and recycling of channels in the endocytic pathway. BecausePCSK9 increased ENaC degradation, it seemed likely thatPCSK9would reduce the pool of ENaC available for exocytosis.To test this possibility, we used a functional strategy wereported previously (49). We covalently modified the cell sur-face pool of ENaC and thenmeasured the rate of appearance of

unmodified channels at the cell surface. For these experiments,we placed a cysteine in the pore of �ENaC (G536C) (43, 49).When the mutant � subunit was coexpressed in FRT epitheliawith wild-type �- and �ENaC,MTSET irreversibly blocked thechannel by modifying the introduced cysteine, as shown in therepresentative current traces in Fig. 5, A and B. BecauseMTSET is not membrane-permeable, the intracellular ENaCpool was protected from modification. Following removal ofMTSET from the bathing solution, wemeasured the increase inENaC current over time as an assay of exocytosis of unmodi-fied/unblocked channels. Fig. 5C shows the averaged timecourses of current recovery, and the time constants are showninD. PCSK9 reduced the maximal increase in current recoveryover time but did not significantly alter the time course of theincrease, as reflected by the lack of difference in the rate con-stant. These results are consistentwith a reduction in the size ofthe ENaC pool available for exocytosis. However, the observeddecrease in current recovery could also be explained by anincrease in the rate of ENaC endocytosis. We therefore testedthe effect of PCSK9 on ENaC endocytosis.

FIGURE 3. PCSK9 reduces ENaC cell surface expression. A, immunoblot analyses of ENaC in the cell surface biotinylated fraction (top panel) and in thetotal cell lysate (bottom panel) from HEK 293 cells transfected with �-, �-, and �ENaC (1 �g each, one subunit contained FLAG epitope) with or withoutPCSK9 (3 �g). Total cDNA was kept constant using GFP cDNA. ENaC protein in �PCSK9 group relative to -PCSK9 group is quantified by densitometry inB (mean � S.E.; n � 3–5; *, p � 0.03). C, immunoblots of Nedd4-2-HA (anti-HA) and �-actin in HEK 293 cells transfected with Nedd4-2-HA (3 �g) with orwithout PCSK9 (3 �g). D, amiloride-sensitive current in Xenopus oocytes expressing human �- and �ENaC with wild-type or mutant �ENaC (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�Y644AENaC-FLAG coexpressed with �Y620A- and �Y627AENaC (1 �g each) with PCSK9 or GFP (3 �g). F, immunoblot of biotinylated (top panel) and total(bottom panel) �ENaC-FLAG coexpressed in HEK 293 cells with �- and �ENaC (1 �g each) with GFP or PCSK9 (wild type or S386A, 3 �g). Irrelevant laneswere removed digitally.

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PCSK9 Does Not Alter Trafficking of the Cell Surface ENaCPool—In Fig. 6, we quantitated endocytosis of mature proteo-lytically cleaved ENaC using a method that we described previ-ously (44). We mutated the two furin consensus sites in theextracellular domain of �ENaC to prevent cleavage by furin(�Cl-2) and then treated the cells briefly with trypsin to generatea pool of proteolytically cleaved channels (65-kDa band). Afterremoval of trypsin, we incubated the cells at 37 °C for 0–60minto allow endocytosis of the cleaved channels and then biotiny-lated and detected channels remaining at the cell surface. Thedisappearance of the 65-kDa band reflects the rate of ENaCendocytosis. Because newly synthesized mutant channelsreaching the cell surface are uncleaved, they can be distin-guished from the cleaved channels undergoing endocytosis. Asshown in Fig. 6, A and B, the 65-kDa cleaved band was rapidlyremoved from the cell surface with a half-life of �15 min, con-sistent with our prior work (44). However, PCSK9 did not alterthe rate of ENaC disappearance from the cell surface. Thus,PCSK9 does not alter ENaC surface expression through achange in endocytosis.Following endocytosis, ENaC can either recycle back to the

cell surface or traffic to lysosomes for degradation. To testwhether PCSK9 alters ENaC surface expression in part by reg-ulating this sorting step, we measured the effect of PCSK9 ondegradation of the cell surface pool of ENaC. In HEK 293 cellsexpressing ENaC, we pulse-labeled the cell surface fraction ofchannels with biotin, incubated the cells at 37 °C for 0–120min, and then quantitated the remaining (non-degraded) bioti-nylated channels. PCSK9 did not increase the rate of degrada-tion of biotinylated �ENaC (Fig. 7, A and B). Rather, it slightlydelayed degradation, which may partially counter the effect ofPCSK9 on ENaC degradation in the biosynthetic pathway.

DISCUSSION

Recent work has focused on the regulation of epithelial Na�

transport by proteases, including furin, a member of the pro-protein convertase family (9). Here we found that ENaC is reg-ulated by PCSK9, another member of this protease family.However, furin and PCSK9 have opposite effects on ENaC cur-rent and they regulate ENaC through different mechanisms. Incontrast to furin, which activates ENaC by proteolytic cleavageof the extracellular domains of the � and � subunits, PCSK9inhibits ENaC by reducing its cell surface expression. More-over, unlike furin, PCSK9 regulates ENaC independent of itsprotease activity.The data indicate that PCSK9 reduces ENaC surface expres-

sion primarily by increasing its degradation in the biosyntheticpathway, which reduces the pool of ENaC available for exocy-tosis. Consistent with this concept, we found that PCSK9decreased ENaC exocytosis and increased the rate of ENaCdegradation in the proteasome.Moreover, PCSK9 had no effecton the rate of ENaC endocytosis or its degradation in the endo-cytic pathway. Coimmunoprecipitation studies suggest thatENaC and PCSK9 interact with one another in their immature(uncleaved) states, likely prior to ENaC cleavage in the Golgiapparatus (althoughwe cannot exclude the possibility that pro-teolytic cleavage induces conformation changes that prohibitbinding). Together, these findings are most consistent with amodel in which PCSK9 enhances endoplasmic reticulum asso-ciated 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 traffick-ing, culminating in increased degradation. Likewise, both occurindependent of PCSK9 protease activity. However, there arealso important differences. Contrary to regulation of ENaC inthe biosynthetic pathway, PCSK9 predominately regulates theLDLR in the endocytic pathway (34, 35). This suggests thatPCSK9 can function in multiple cellular compartments. Thereare also differences in themechanisms of binding. PCSK9 inter-acts with the LDLR through two interfaces. Crystallizationrevealed that the catalytic domain of PCSK9 binds to the epi-dermal growth factor-like A domain in the LDLR (36). A recentreport found a second interaction between the PCSK9C-termi-nal domain and the LDLR ligand binding domain, which wasrequired for regulation (52). Importantly, ENaC lacks homolo-gous motifs, indicating that it interacts with PCSK9 through anovel binding mechanism. This interaction could be direct orcould occur indirectly through an adaptor protein.The proprotein convertase furin regulates ENaC by proteo-

lytic cleavage of the extracellular domains of �- and �ENaC,which releases inhibitory peptides (9). In this manner, furinregulates ENaC gating, converting near-silent channels intoactive channels. However, there may be an additional level ofcomplexity. Furin also proteolytically cleaves PCSK9, whichinactivates it (53). Thus, through a decrease in PCSK9 activity,furin could increase ENaC cell surface expression. This raisesthe interesting possibility that furin regulates ENaC throughdual effects on channel trafficking and gating.

FIGURE 4. PCSK9 increases ENaC degradation. A, immunoblot analyses of�ENaC-FLAG in HEK 293 cells cotransfected with �- and �ENaC (1 �g each)with or without PCSK9 (3 �g). The cell were treated with cycloheximide (CHX)(10 �g/ml) for 0 –120 min prior to lysis. B, quantification �ENaC, relative to 0min time point (mean � S.E.; n � 6; *, p � 0.007). C, immunoblot analyses of�ENaC-FLAG in HEK 293 cells cotransfected with �- and �ENaC (1 �g each)with or without PCSK9 (3 �g). The cells were treated with N-acetyl-Leu-Leu-norleucinal (ALLN) (10 �M), NH4Cl (5 mM), or vehicle for 2 h prior to lysis. Dataare representative of at least three experiments.

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Defects in ENaC regulation are responsible for the majorityof the known genetic forms of hypertension, which is an impor-tant risk factor for coronary heart disease and other cardiovas-cular diseases. Thus, PCSK9 could modulate cardiovascularrisk in part through its regulation of ENaC.We speculate that adecrease in PCSK9 activity would increase renal Na� absorp-tion and, therefore, raise the risk of hypertension and associatedcardiovascular disease. However, such a mechanism wouldcounter the previously reported effect of PCSK9 mutations oncardiovascular risk. Activating mutations were found toincrease the risk, whereas loss-of-function mutations reducedthe risk (29–33). These effects are thought to occur throughchanges in expression of the LDLR, which produce changes inserum levels of cholesterol. Thus, it is possible that PCSK9 reg-

ulation of ENaC and the LDLR have opposing effects on cardio-vascular risk. On the other hand, our data suggest that PCSK9regulates ENaC and the LDLR through different binding sitesand different mechanisms. Thus, the PCSK9 mutations thatdisrupt LDLR regulation may have dissimilar effects on ENaC.Additional work will be required to test whether naturallyoccurring mutations in PCSK9 alter ENaC trafficking, renalNa� 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 reagentsand DNA sequencing.

FIGURE 5. PCSK9 decreases ENaC exocytosis. Short-circuit currents were recorded in FRT epithelia transfected with �-, �-, and �G536CENaC subunits (0.16 �geach) with or without PCSK9 (0.5 �g). 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 ofthe data in C (mean � S.E.; n � 7; n.s., p � 0.05).

FIGURE 6. PCSK9 does not alter ENaC endocytosis. A, immunoblot analysis(anti-FLAG) of biotinylated�Cl-2 ENaC-FLAG in HEK 293 cells cotransfected with�-and �ENaC (1 �g each) with or without PCSK9 (3 �g). The cells were treated with5 �g/ml trypsin for 5 min, incubated at 37 °C for 0–60 min, and then biotinylated.B, quantification of the cleaved �ENaC band relative to 0 min (mean�S.E., n �5).

FIGURE 7. PCSK9 does not alter degradation of cell surface ENaC. A, immu-noblots (anti-FLAG) of biotinylated �ENaC in HEK 293 cells transfected with�ENaC-FLAG, �ENaC, and �ENaC (1 �g each) with or without PCSK9 (3 �g).Cell surface proteins were pulse-labeled with biotin and then the cells wereincubated at 37 °C for 0 –120 min. B, biotinylated �ENaC at each time wasquantified relative to 0 min (mean � S.E.; n � 3; *, p � 0.05).

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Vikas Sharotri, Daniel M. Collier, Diane R. Olson, Ruifeng Zhou and Peter M. SnyderSubtilisin/Kexin Type 9 (PCSK9)

Regulation of Epithelial Sodium Channel Trafficking by Proprotein Convertase

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