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Paraoxonase 3 functions as a chaperone to decrease functional expression of the epithelial sodium channel

Open AccessPublished:February 20, 2020DOI:https://doi.org/10.1074/jbc.RA119.011789
      The paraoxonase (PON) family comprises three highly conserved members: PON1, PON2, and PON3. They are orthologs of Caenorhabditis elegans MEC-6, an endoplasmic reticulum–resident chaperone that has a critical role in proper assembly and surface expression of the touch-sensing degenerin channel in nematodes. We have shown recently that MEC-6 and PON2 negatively regulate functional expression of the epithelial Na+ channel (ENaC), suggesting that the chaperone function is conserved within this family. We hypothesized that other PON family members also modulate ion channel expression. Pon3 is specifically expressed in the aldosterone-sensitive distal tubules in the mouse kidney. We found here that knocking down endogenous Pon3 in mouse cortical collecting duct cells enhanced Na+ transport, which was associated with increased γENaC abundance. We further examined Pon3 regulation of ENaC in two heterologous expression systems, Fisher rat thyroid cells and Xenopus oocytes. Pon3 coimmunoprecipitated with each of the three ENaC subunits in Fisher rat thyroid cells. As a result of this interaction, the whole-cell and surface abundance of ENaC α and γ subunits was reduced by Pon3. When expressed in oocytes, Pon3 inhibited ENaC-mediated amiloride-sensitive Na+ currents, in part by reducing the surface expression of ENaC. In contrast, Pon3 did not alter the response of ENaC to chymotrypsin-mediated proteolytic activation or [2-(trimethylammonium)ethyl]methanethiosulfonate–induced activation of αβS518Cγ, suggesting that Pon3 does not affect channel open probability. Together, our results suggest that PON3 regulates ENaC expression by inhibiting its biogenesis and/or trafficking.

      Introduction

      The epithelial Na+ channel (ENaC)
      The abbreviations used are: ENaC
      epithelial Na+ channel
      BP
      blood pressure
      NCC
      Na–Cl cotransporter
      PON
      paraoxonase
      KD
      knockdown
      FRT
      Fisher rat thyroid
      DCT
      distal convoluted tubule
      CNT
      connecting tubule
      CD
      collecting duct
      PC
      principal cell
      IC
      intercalated cell
      V-ATPase
      vacuolar H+-ATPase
      Isc
      short circuit current
      NC
      negative control
      γGT
      γ-glutamyl transferase
      ROMK
      renal outer medullary K+ channel
      MTSET
      [2-(trimethylammonium)ethyl]methanethiosulfonate
      ANOVA
      analysis of variance
      mCCD
      mouse cortical collecting duct
      PO
      open probability.
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      ). Misfolded subunits are subjected to endoplasmic reticulum–associated degradation via the proteasome (
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      ). The retrieved channel complex may then be shuttled to the lysosome for degradation or deubiquitinated and recycled back to the cell surface (
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      Trafficking and cell surface stability of ENaC.
      ). Together, this multistage machinery determines the total number of functional channels at the cell surface. Molecular chaperones have been implicated in multiple key steps during ENaC biogenesis, trafficking, and degradation (
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      ), including members of the paraoxonase (PON) family.
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      ), they differ in their tissues expression and substrate specificity. PON2 is ubiquitously expressed in various tissues and most major organs, whereas PON1 and PON3 were thought to be mainly synthesized by the liver and circulate within high-density lipoprotein particles (
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      ,
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      ). However, recent studies have detected PON3 in epithelial cells within many tissues, including the digestive tract, respiratory system, reproductive system, and kidneys (
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      ). Although all three PONs possess lactonase activity, only PON1 can hydrolyze organophosphates, such as paraoxon and its derivatives (
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      ). PONs share evolutionary conservation with Caenorhabditis elegans MEC-6 and several other predicted proteins in nematodes. MEC-6 is a chaperone protein that is required for proper folding, assembly, and surface expression of the touch-sensitive MEC-4/MEC-10 channel in the nematode's touch receptor neurons (
      • Chelur D.S.
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      ,
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      Caenorhabditis elegans paraoxonase-like proteins control the functional expression of DEG/ENaC mechanosensory proteins.
      ). We have shown recently that PON2 inhibits ENaC activity by reducing channel surface expression (
      • Shi S.
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      Regulation of the epithelial Na+ channel by paraoxonase-2.
      ), suggesting that the chaperone function is conserved between mammalian PONs and their nematode orthologs.
      There is evidence suggesting that mammalian PONs have important roles in kidney function. Pon1 KO mice were hypotensive with a lower serum aldosterone level (
      • Gamliel-Lazarovich A.
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      • Vaya J.
      • Aviram M.
      • Keidar S.
      Paraoxonase1 deficiency in mice is associated with hypotension and increased levels of 5,6-epoxyeicosatrienoic acid.
      ). Knockdown (KD) of Pon2 by renal subcapsular infusion of Pon2 siRNA led to hypertension in rats (
      • Yang Y.
      • Zhang Y.
      • Cuevas S.
      • Villar V.A.
      • Escano C.
      • D Asico L.
      • Yu P.
      • Grandy D.K.
      • Felder R.A.
      • Armando I.
      • Jose P.A.
      Paraoxonase 2 decreases renal reactive oxygen species production, lowers blood pressure, and mediates dopamine D2 receptor-induced inhibition of NADPH oxidase.
      ). Although it is unclear whether altered ENaC activity and/or expression contribute to the changes in BP in these models, they collectively support a role of PONs in maintaining normal BP. Being the most neglected member of this family, the role of PON3 in BP control has not been investigated in whole-animal studies. However, Pon3 transcripts have been detected in multiple nephron segments in the rat kidney and in principal cells and intercalated cells of the mouse distal nephron (
      • Chen L.
      • Lee J.W.
      • Chou C.L.
      • Nair A.V.
      • Battistone M.A.
      • Păunescu T.G.
      • Merkulova M.
      • Breton S.
      • Verlander J.W.
      • Wall S.M.
      • Brown D.
      • Burg M.B.
      • Knepper M.A.
      Transcriptomes of major renal collecting duct cell types in mouse identified by single-cell RNA-seq.
      ,
      • Lee J.W.
      • Chou C.L.
      • Knepper M.A.
      Deep sequencing in microdissected renal tubules identifies nephron segment-specific transcriptomes.
      ). PON3 shares high sequence homology with PON2 (>60%), which led us to hypothesize that PON3 also functions as a chaperone to regulate ENaC expression. This study examined the expression of Pon3 in the mouse kidney and its effect on ENaC functional expression by silencing endogenous Pon3 in mouse cortical collecting duct cells or overexpressing Pon3 in cultured FRT cells or Xenopus oocytes.

      Results

      Pon3 expression in the mouse kidney

      To determine whether ENaC is an endogenous substrate for PON3, we first asked whether PON3 localizes to the aldosterone-sensitive distal nephron where ENaC resides. Despite the detection of Pon3 transcripts in multiple nephron segments and cell types in rodents (
      • Chen L.
      • Lee J.W.
      • Chou C.L.
      • Nair A.V.
      • Battistone M.A.
      • Păunescu T.G.
      • Merkulova M.
      • Breton S.
      • Verlander J.W.
      • Wall S.M.
      • Brown D.
      • Burg M.B.
      • Knepper M.A.
      Transcriptomes of major renal collecting duct cell types in mouse identified by single-cell RNA-seq.
      ,
      • Lee J.W.
      • Chou C.L.
      • Knepper M.A.
      Deep sequencing in microdissected renal tubules identifies nephron segment-specific transcriptomes.
      ), the expression of PON3 protein in the kidney has not been thoroughly investigated. We approached this question with immunofluorescence staining. Kidney sections from WT C57BL/6 mice were incubated with an anti-PON3 antibody and imaged from the cortex to the medulla using a tile scan technique. As shown in the automatically merged tile scans (Fig. 1A), Pon3 localizes mainly in tubular epithelial cells within the cortex but is also present in a specific population of cells of the medulla. To further define the tubular expression of Pon3, we performed costaining experiments using specific markers of different nephron segments. As shown in the overlaid images (Fig. 1B), Pon3 was absent in tubules showing positive staining of the Na–K–Cl cotransporter or parvalbumin, suggesting that Pon3 is not expressed in the thick ascending limb or the early distal convoluted tubule (DCT1 (
      • Plotkin M.D.
      • Kaplan M.R.
      • Verlander J.W.
      • Lee W.S.
      • Brown D.
      • Poch E.
      • Gullans S.R.
      • Hebert S.C.
      Localization of the thiazide sensitive Na-Cl cotransporter, rTSC1 in the rat kidney.
      ,
      • Debonneville C.
      • Flores S.Y.
      • Kamynina E.
      • Plant P.J.
      • Tauxe C.
      • Thomas M.A.
      • Münster C.
      • Chraïbi A.
      • Pratt J.H.
      • Horisberger J.D.
      • Pearce D.
      • Loffing J.
      • Staub O.
      Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na+ channel cell surface expression.
      )). In contrast, we detected Pon3 in a subpopulation of NCC-positive tubules, likely to be the late DCT (DCT2, Fig. 1B). Of a total of 148 NCC-positive tubules, 91 tubules showed positive staining for Pon3. Within these NCC/Pon3 double-positive tubules, Pon3 was ubiquitously expressed in nearly all NCC-positive cells. As shown in Fig. 1B, Pon3 also colocalized with the cytoplasmic Ca2+-binding protein calbindin D28K (CaBP28K), a marker for distal tubules with higher abundance in the DCT2 and connecting tubule (CNT) and lower expression in the DCT1 and collecting duct (CD) (
      • Taylor A.N.
      • McIntosh J.E.
      • Bourdeau J.E.
      Immunocytochemical localization of vitamin D-dependent calcium-binding protein in renal tubules of rabbit, rat, and chick.
      ,
      • Loffing J.
      • Loffing-Cueni D.
      • Valderrabano V.
      • Kläusli L.
      • Hebert S.C.
      • Rossier B.C.
      • Hoenderop J.G.
      • Bindels R.J.
      • Kaissling B.
      Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron.
      ). Among 78 distal tubules with strong CaBP28K staining, 90.6% ± 5.3% cells expressed both CaBP28K and Pon3. To further examine in which cell type Pon3 is expressed, WT mouse kidney sections were costained for Pon3 with the principal cell (PC) marker aquaporin 2 (AQP2) or the intercalated cell (IC) marker vacuolar H+-ATPase (V-ATPase) (
      • Fushimi K.
      • Uchida S.
      • Hara Y.
      • Hirata Y.
      • Marumo F.
      • Sasaki S.
      Cloning and expression of apical membrane water channel of rat kidney collecting tubule.
      ,
      • Miller R.L.
      • Zhang P.
      • Smith M.
      • Beaulieu V.
      • Paunescu T.G.
      • Brown D.
      • Breton S.
      • Nelson R.D.
      V-ATPase B1-subunit promoter drives expression of EGFP in intercalated cells of kidney, clear cells of epididymis and airway cells of lung in transgenic mice.
      ). As shown in the merged images in Fig. 1B, Pon3 was expressed in both PCs and ICs but with distinctive expression patterns. Nearly all ICs within a total of 97 V-ATPase–positive tubules were also positive for Pon3. Although all AQP2-positive tubules (n = 115) exhibited Pon3 staining, only 25.1% ± 8.8% PCs within AQP2/Pon3 double-positive tubules expressed both AQP2 and Pon3. The specificity of the anti-PON3 antibody was validated with kidney sections of Pon3 KO mice. We did not observe significant Pon3 staining in tubules from KO animals and only weak staining within glomeruli (Fig. 1C). Together, our data suggest that Pon3 is primarily expressed in the DCT2, CNT, and CD of the aldosterone-sensitive distal nephron.
      Figure thumbnail gr1
      Figure 1Nephron segment–specific expression of Pon3 in the mouse kidney. A, transverse-sectioned kidneys of WT C57BL6 mice were stained for PON3 (green) and imaged from the cortex to the medulla using a tile scan technique. Scale bar = 100 μm. B, merged images of kidney sections costained for PON3 (green) and nephron segment–specific markers (red) in WT mice: NKCC2, Na-K-Cl cotransporter PVALB, parvalbumin. C, anti-PON3 antibody specificity was validated in Pon3 KO mice. Scale bars in B and C = 20 μm. Representative images are shown for kidney sections obtained from four WT mice or three Pon3 KO mice.

      Pon3 KD in mouse CCD (mCCD) cells enhances ENaC-mediated Na+ transport

      As Pon3 is expressed in principal cells of the distal nephron (Fig. 1B), we examined the role of endogenous Pon3 in regulating ENaC expression and function with siRNA-mediated KD. mCCD cells, derived from the mouse cortical CD, express all three ENaC subunits as well as mineralocorticoid and glucocorticoids receptors (
      • Bens M.
      • Vallet V.
      • Cluzeaud F.
      • Pascual-Letallec L.
      • Kahn A.
      • Rafestin-Oblin M.E.
      • Rossier B.C.
      • Vandewalle A.
      Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line.
      ). As expected, endogenous Pon3 was detected as a single band near 40 kDa in mCCD cells (Fig. 2C). To knock down Pon3, mCCD cells were transiently transfected with Pon3-specific siRNA, and ENaC-mediated Na+ transport was determined with short-circuit current (Isc) measurements. As shown in Fig. 2, Pon3 expression in mCCD cells was reduced 55% ± 14% (n = 11, p < 0.001) compared with negative control (NC) cells transfected with scrambled siRNAs (Fig. 2D). As a result, we observed a 1.2 ± 0.2-fold (n = 12, p < 0.01) increase in amiloride-sensitive Isc in Pon3 KD cells (Fig. 2B) without noticeable changes in transepithelial resistance (1.13 ± 0.49 kilo-ohm for Pon3 KD cells versus 1.22 ± 0.34 kilo-ohm for NC cells, n = 12, p = 0.62). The enhanced Na+ transport in Pon3 KD cells was associated with a 1.4 ± 0.4-fold increase in the abundance of cleaved γENaC (n = 11, p < 0.05). The abundance of uncleaved γENaC was not affected by Pon3 KD (Fig. 2E). In addition, the ratio of cleaved versus total γENaC was significantly higher in Pon3 KD cells (Fig. 2F), suggesting enhanced channel proteolytic processing. Together, our data suggest that Pon3 regulates endogenous ENaC functional expression in mCCD cells.
      Figure thumbnail gr2
      Figure 2Pon3 KD in mCCD cells enhances the functional expression of endogenous ENaC. mCCD cells were transfected with Pon3-specific siRNA (Pon3 KD) or scrambled siRNA as an NC. Transfected cells were then cultured on permeable filters, and amiloride (Amil)-sensitive Isc were determined with an Ussing chamber assay. A, representative traces in NC cells (black) or Pon3 KD cells (red). After reaching a steady current, 10 μm amiloride (black bar) was added to the apical side of the Ussing chamber to block ENaC. B, summary of the amiloride-sensitive Isc of Pon3 KD cells normalized (Norm.) to the average Isc of NC cells from the same experiment. C, following the recordings, whole-cell lysates were collected from the filters to blot for γENaC, Pon3, and actin as a loading control. The asterisk and arrowhead indicate uncleaved and cleaved γENaC, respectively. D and E, whole-cell expression of Pon3 (D) and γENaC (E) were normalized to actin and are shown as a percentage of NC cells. F, to access the extent of γENaC cleavage, the ratio of the cleaved γENaC to total γENaC (cleaved and uncleaved) was estimated for each sample and is shown as a percentage of NC cells. Experiments were repeated a total of four times with three individual transfections of scrambled siRNA or Pon3 siRNA for each experiment. Data are shown in scatter-dot plots, with a horizontal bar indicating the mean. Statistical comparisons were analyzed with a nonparametric Mann–Whitney test or with one-way ANOVA followed by Sidak's multiple comparisons test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

      Pon3 interacts with ENaC subunits

      We then performed coimmunoprecipitation to investigate whether PON3 forms a complex with ENaC subunits. Briefly, we transfected FRT cells with mouse Pon3 that had a C-terminal Myc-FLAG epitope tag, mouse ENaC, or both. In each case, only one ENaC subunit had an N-terminal HA and a C-terminal V5 epitope tag (HAαV5βγ, αHAβV5γ, or αβHAγV5). Whole-cell lysates were either precipitated with anti-V5 antibodies to pulldown ENaC and probe for Pon3 (Fig. 3A) or precipitated with anti-FLAG antibodies to pulldown Pon3 and probe for ENaC subunits (Fig. 3B). As shown in Fig. 3A, Pon3-Myc-FLAG (∼45 kDa) was detected in V5 precipitates of FRT cells coexpressing Pon3 and ENaC but not in cells transfected with only Pon3. Vice versa, ENaC subunits could be detected only in FLAG precipitates of FRT cells coexpressing Pon3 and ENaC but not in cells transfected with ENaC alone (Fig. 3B). Expression of Pon3 or ENaC subunits was detected in whole-cell lysate as expected (input). Our data suggest that Pon3 specifically interacts with ENaC subunits when coexpressed in FRT cells.
      Figure thumbnail gr3
      Figure 3Pon3 interacts with ENaC subunits in FRT cells. FRT cells were transiently transfected with cDNAs encoding mouse Pon3 and/or ENaC subunits. In each case, only one ENaC subunit had a C-terminal V5, tag as indicated above. Pon3 was cloned in the pCMV6 vector, which includes a C-terminal Myc-FLAG tag. A, whole-cell lysates were immunoprecipitated (IP) with anti-V5 antibodies to pull down ENaC and blotted for Pon3. B, vice versa, the lysates were immunoprecipitated with anti-FLAG antibodies to pull down Pon3 and blotted for ENaC subunits with V5 antibodies. The anti-FLAG antibody and the V5 antibody were raised in mice. The arrow indicates the heavy chain of the anti-FLAG antibody recognized by the secondary antibody. Expression of ENaC and Pon3 was also assessed in whole-cell lysates (input). Vertical dashed lines indicate that the same blot of different exposures is shown for immunoprecipitate and input. The mobility of the molecular mass standards (kilodaltons) is shown. Experiments were repeated three to for times for each condition.

      Pon3 reduces ENaC expression in FRT cells

      As ENaC functional expression was enhanced by Pon3 KD in mCCD cells, we examined whether overexpressing Pon3 affects ENaC expression in FRT cells. We cotransfected FRT cells with ENaC (HAαV5βγ) with either the Pon3 plasmid or an equal amount of the empty vector. Surface proteins were biotinylated on ice and recovered with NeutrAvidin beads. The expression of ENaC α and γ subunits was assessed in surface precipitates and whole-cell lysates. GAPDH was absent in surface precipitates, indicating that the surface fractions were free of contamination from intracellular proteins (Fig. 4A, bottom). In FRT cells coexpressing Pon3, whole-cell ENaC expression was significantly lower for full-length αENaC (48% ± 22%, n = 5–6, p < 0.01), the cleaved αENaC (64% ± 14%, n = 5–6, p < 0.001) and γENaC (41% ± 13%, n = 5–6, p < 0.01, Fig. 4B). In contrast, Pon3 did not alter the ratio of surface abundance versus whole-cell abundance for αENaC or γENaC (Fig. 4C), suggesting that Pon3 not only reduced ENaC subunit whole-cell expression but also decreased channel density at the cell surface in FRT cells. This inhibitory effect of Pon3 is consistent with our findings of the effects of endogenous Pon3 on ENaC in mCCD cells (Fig. 2). As a control, we tested whether γ-glutamyl transferase (γGT), an unrelated type II protein affects ENaC expression in FRT cells. Our data suggest that coexpressing mouse γGT has no effect on ENaC abundance at the whole-cell level in FRT cells (Fig. 4, D and E).
      Figure thumbnail gr4
      Figure 4Pon3 reduces ENaC surface and whole-cell expression in FRT cells. A, FRT cells were transiently transfected with three ENaC subunits, where only the α subunit had an N-terminal HA tag and a C-terminal V5 tag. Equal amounts of the mouse Pon3 plasmid or the pCMV6 vector were cotransfected. Surface ENaC was labeled with biotin and recovered with NeutrAvidin beads at 4 °C. Blots were probed for the α subunit, γ subunit, Pon3, or GAPDH in the biotinylated surface fraction and in 5% of the total whole-cell lysates. B, the abundance of the α subunit (full-length 95 kDa and cleaved 30 kDa, indicated by arrowheads) or the γ subunit (∼75 kDa) in whole-cell lysate was normalized to the loading control (GAPDH) and expressed as the percentage in cells transfected with ENaC alone (−Pon3). C, the abundance of the α subunit (full-length 95 kDa and cleaved 30 kDa) or the γ subunit (∼75 kDa) at the cell surface was normalized to whole-cell abundance of the α or γ subunit, respectively, and then expressed as the percentage in cells transfected with ENaC alone (−Pon3). D, as a control, FRT cells were cotransfected with HAαV5βγ and with either γ-glutamyl transferase (+γGT) or an equal amount of the pCDNA3 vector (−γGT). Whole-cell lysates were collected and probed for both αENaC and γGT (indicated by the arrow). E, the abundance of the α subunit (95 kDa and 30 kDa) was normalized to the loading control (GAPDH) and expressed as the percentage in cells transfected with ENaC alone (−γGT). Experiments were repeated a total of three times with FRT cells of different passages. The summarized data are shown in a scatter-dot plot, with a horizontal bar indicating the mean. Statistical comparisons were analyzed with one-way ANOVA followed by Sidak's multiple comparisons test (**, p < 0.01; ***, p < 0.001).

      Pon3 inhibits ENaC activity by reducing channel surface expression in Xenopus oocytes

      To further investigate the role of Pon3 in regulating ENaC functional expression, we injected mature oocytes with cRNA mixtures encoding WT ENaC alone or with an equal amount of mouse Pon3 cRNA. Whole-cell Na+ currents were measured 24–30 h after injection by clamping oocytes at −100 mV (Fig. 5A). The average amiloride-sensitive Na+ current in oocytes coexpressing ENaC and Pon3 (−1.4 ± 1.4 μA, n = 60) is ∼40% lower than in oocytes expressing ENaC alone (−2.4 ± 1.8 μA, n = 62, p < 0.01). This is similar to the extent of inhibition of PON2 on ENaC activity (
      • Shi S.
      • Buck T.M.
      • Kinlough C.L.
      • Marciszyn A.L.
      • Hughey R.P.
      • Chalfie M.
      • Brodsky J.L.
      • Kleyman T.R.
      Regulation of the epithelial Na+ channel by paraoxonase-2.
      ). The inhibitory effect of Pon3 is specific to ENaC, as Pon3 did not alter renal outer medullary K+ (Romk) channel activity (−2.5 ± 1.8 μA in oocytes expressing Romk versus −2.2 ± 1.9 μA in oocytes coexpressing Pon3 and Romk, Fig. 5C).
      Figure thumbnail gr5
      Figure 5Pon3 inhibits ENaC activity in oocytes. A, representative traces of whole-cell Na+ currents measured in oocytes expressing mouse ENaC alone (black) or coexpressing Pon3 (+Pon3, red), with the holding potential set at −100 mV. After reaching a steady current, 10 μm amiloride (Amil, black bar) was added to the bath to block ENaC. B, summary of the amiloride-sensitive Na+ currents in oocytes expressing ENaC or ENaC and Pon3. C, summary of the BaCl2-sensitive K+ currents of oocytes injected with Romk with or without Pon3. Experiments were repeated with four batches of oocytes, and the pooled data are shown in scatter-dot plots, with a horizontal bar indicating the mean. The number of oocytes assessed for each condition is listed. Statistical comparisons were analyzed with nonparametric Mann–Whitney tests (**, p < 0.01).
      The inhibitory effect of Pon3 on ENaC activity suggests a reduction in channel open probability (PO) and/or in the number of functional channels at the cell surface. As Pon3 reduced ENaC surface expression in FRT cells (Fig. 4), we examined the effect of Pon3 on channel surface expression in oocytes using a FLAG epitope–tagged ENaC β subunit, as described previously (
      • Carattino M.D.
      • Hill W.G.
      • Kleyman T.R.
      Arachidonic acid regulates surface expression of epithelial sodium channels.
      ). As shown in Fig. 6A, the surface expression of the nontagged channel was 0.2 ± 0.5 × 106 (shown as relative light units, n = 21) but rose to 1.6 ± 1.8 × 107 in oocytes expressing FLAG-tagged ENaC (n = 33, p < 0.0001). Notably, this value was significantly reduced in oocytes coexpressing Pon3 (3.3 ± 3.3 × 106, n = 36, p < 0.01). In contrast, whole-cell expression of βENaC in oocytes was not altered by Pon3 (Fig. 6C).
      Figure thumbnail gr6
      Figure 6Pon3 reduces ENaC surface expression in oocytes. A, a chemiluminescence assay was performed to assess the surface abundance of ENaC in oocytes expressing WT (nontagged) ENaC, ENaC with a β subunit bearing an extracellular FLAG tag (ENaC-FLAG), or ENaC-FLAG coexpressed with mouse Pon3. Relative light units measured for individual oocytes are shown. Experiments were repeated with oocytes harvested from three individual frogs. The number of oocytes assessed for each group is listed. Data are shown in a scatter-dot plot, with horizontal bars indicating the means. Statistical comparisons were analyzed with nonparametric Kruskal–Wallis test followed by Dunn's multiple comparisons test (**, p < 0.01; ****, p < 0.0001). B, βENaC was probed in whole-cell homogenates of oocytes expressing ENaC or ENaC and Pon3 with the StressMarq β antibody. Noninjected oocytes from the same batch were used as a negative control, and GAPDH was probed as a loading control. C, the expression of βENaC in the presence or absence of Pon3 was normalized to GAPDH and then expressed as the percentage in oocytes expressing ENaC only (−Pon3) for individual experiments. Data are shown in a scatter-dot plot, with a horizontal bar indicating the mean. Experiments were repeated with six different batches of oocytes. No statistically significant difference was observed when data were analyzed with a nonparametric Wilcoxon signed-rank test.

      Pon3 does not alter ENaC PO in Xenopus oocytes

      ENaC is gated in response to external cues, such as proteolytic cleavage, extracellular Na+, and laminar shear stress, that primarily modulate channel PO (
      • Shi S.
      • Carattino M.D.
      • Hughey R.P.
      • Kleyman T.R.
      ENaC regulation by proteases and shear stress.
      ,
      • Kleyman T.R.
      • Kashlan O.B.
      • Hughey R.P.
      Epithelial Na+ channel regulation by extracellular and intracellular factors.
      ). Specific proteases activate ENaC by releasing the embedded inhibitory tracts from the α or γ subunit, increasing channel PO (
      • Carattino M.D.
      • Sheng S.
      • Bruns J.B.
      • Pilewski J.M.
      • Hughey R.P.
      • Kleyman T.R.
      The epithelial Na+ channel is inhibited by a peptide derived from proteolytic processing of its α subunit.
      ,
      • Hughey R.P.
      • Bruns J.B.
      • Kinlough C.L.
      • Harkleroad K.L.
      • Tong Q.
      • Carattino M.D.
      • Johnson J.P.
      • Stockand J.D.
      • Kleyman T.R.
      Epithelial sodium channels are activated by furin-dependent proteolysis.
      ,
      • 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.
      ,
      • 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.
      ,
      • Passero C.J.
      • Mueller G.M.
      • Myerburg M.M.
      • Carattino M.D.
      • Hughey R.P.
      • Kleyman T.R.
      TMPRSS4-dependent activation of the epithelial sodium channel requires cleavage of the γ-subunit distal to the furin cleavage site.
      ). We examined whether Pon3 affects channel activation by a protease, α-chymotrypsin. Oocytes were treated with α-chymotrypsin (2 μg/ml) for 2 min until currents reached a steady state (Fig. 7A). If Pon3 reduces channel PO, we predicted that the fold increase in ENaC current following α-chymotrypsin treatment would be altered. However, we found that chymotrypsin elicited a 1.9 ± 1.0-fold increase in whole-cell Na+ currents in oocytes expressing ENaC and a similar 1.9 ± 0.5-fold increase in oocytes coexpressing ENaC and Pon3 (Fig. 7C). As a result, the post-α-chymotrypsin treatment currents were still lower in the presence of Pon3 (−2.7 ± 1.5 μA, n = 21, p < 0.0001) compared with oocytes only expressing ENaC (−5.5 ± 2.3 μA, n = 21).
      Figure thumbnail gr7
      Figure 7Pon3 does not alter ENaC activation by α-chymotrypsin. A, superimposed traces from oocytes expressing mouse ENaC (black) or coexpressing mouse Pon3 (red). Oocytes were perfused with α-chymotrypsin (gray bar, 2 μg/ml) for 2 min until the current reached a steady state. Amiloride (Amil) was added to the bath at the beginning and the end of each recording (black bars). B, amiloride-sensitive whole-cell Na+ currents (I_Amil) measured prior to (Ibasal) and following chymotrypsin treatment (Ichymo) are shown for individual oocytes. Statistical comparisons were analyzed with two-way ANOVA followed by Sidak's multiple comparisons test (**, p < 0.01; ****, p < 0.0001). C, the -fold increase of ENaC currents in response to α-chymotrypsin (Ichymo/Ibasal) was estimated for individual oocytes. Data pooled from three experiments are shown in a scatter-dot plot, with the horizontal bar indicating the mean. The number of oocytes assessed for each group is listed. Statistical comparisons were analyzed with a nonparametric Mann–Whitney test.
      To examine the effect of Pon3 on channel PO in a more direct manner, we measured the response of ENaC bearing a Cys mutation at the degenerin site of the β subunit (αβS518Cγ) to [2-(trimethylammonium)ethyl]methanethiosulfonate MTSET. The activity of this channel rises when a disulfide bond is formed between MTSET and the introduced Cys within the channel pore, reflecting an increase in channel PO (
      • Kellenberger S.
      • Gautschi I.
      • Schild L.
      An external site controls closing of the epithelial Na+ channel ENaC.
      ,
      • Snyder P.M.
      • Olson D.R.
      • Bucher D.B.
      A pore segment in DEG/ENaC Na+ channels.
      ). As shown in Fig. 8, the whole-cell Na+ currents in oocytes expressing αβS518Cγ was increased 2.1 ± 0.7-fold by MTSET (n = 16). The presence of Pon3 did not alter the effect of MTSET on ENaC activity, as a 2.0 ± 0.4-fold increase in whole-cell Na+ currents was observed in oocytes coexpressing αβS518Cγ and Pon3 (n = 16, Fig. 8B). Together, our data suggest that Pon3 inhibits ENaC activity in oocytes by reducing channel surface expression and not PO.
      Figure thumbnail gr8
      Figure 8Pon3 does not affect ENaC gating in response to MTSET modification. A, recordings from oocytes expressing mouse αβS518Cγ alone (black) or coexpressing Pon3 (red) are superimposed. Freshly prepared MTSET (1 mm) was applied to oocytes via perfusion to modify the introduced -SH group at the degenerin site of the β subunit (S518C, gray bar). At the beginning and end of each recording, 10 μm amiloride (Amil) was added to the bath to block ENaC activity (black bars). B, the -fold increase of ENaC currents elicited by MTSET (IMTSET/Ibasal) was estimated for individual oocytes harvested from three different frogs. Pooled data are shown in a scatter-dot plot, with the horizontal bar indicating the mean. The number of oocytes assessed for each group is listed. Statistical comparisons were analyzed with a nonparametric Mann–Whitney test.

      Discussion

      PON family members are encoded by highly conserved genes, although the three members have distinct substrate specificities and tissue expression (
      • Devarajan A.
      • Shih D.
      • Reddy S.T.
      Inflammation, infection, cancer and all that: the role of paraoxonases.
      ,
      • Précourt L.P.
      • Amre D.
      • Denis M.C.
      • Lavoie J.C.
      • Delvin E.
      • Seidman E.
      • Levy E.
      The three-gene paraoxonase family: physiologic roles, actions and regulation.
      ,
      • Mackness M.I.
      • Mackness B.
      • Durrington P.N.
      • Connelly P.W.
      • Hegele R.A.
      Paraoxonase: biochemistry, genetics and relationship to plasma lipoproteins.
      ). A recent study by Clark and co-workers (
      • Meyer W.K.
      • Jamison J.
      • Richter R.
      • Woods S.E.
      • Partha R.
      • Kowalczyk A.
      • Kronk C.
      • Chikina M.
      • Bonde R.K.
      • Crocker D.E.
      • Gaspard J.
      • Lanyon J.M.
      • Marsillach J.
      • Furlong C.E.
      • Clark N.L.
      Ancient convergent losses of Paraoxonase 1 yield potential risks for modern marine mammals.
      ) identified convergent functional loss of PON1 but not PON2 or PON3 in marine mammals. PON1 contained lesions within the coding region, rendering marine mammals susceptible to neurotoxicity induced by organophosphorus compounds. In contrast, PON1 was intact in all 53 surveyed terrestrial mammal genomes (
      • Meyer W.K.
      • Jamison J.
      • Richter R.
      • Woods S.E.
      • Partha R.
      • Kowalczyk A.
      • Kronk C.
      • Chikina M.
      • Bonde R.K.
      • Crocker D.E.
      • Gaspard J.
      • Lanyon J.M.
      • Marsillach J.
      • Furlong C.E.
      • Clark N.L.
      Ancient convergent losses of Paraoxonase 1 yield potential risks for modern marine mammals.
      ). Like PON1, PON3 is highly expressed in the gallbladder and liver (
      • Shih D.M.
      • Xia Y.R.
      • Yu J.M.
      • Lusis A.J.
      Temporal and tissue-specific patterns of Pon3 expression in mouse: in situ hybridization analysis.
      ), and Pon3 KO mice exhibit altered metabolism of lipid and bile acids. They are also susceptible to developing obesity, atherosclerosis, and large gallstones when fed a diet containing cholic acid and cholesterol (
      • Shih D.M.
      • Yu J.M.
      • Vergnes L.
      • Dali-Youcef N.
      • Champion M.D.
      • Devarajan A.
      • Zhang P.
      • Castellani L.W.
      • Brindley D.N.
      • Jamey C.
      • Auwerx J.
      • Reddy S.T.
      • Ford D.A.
      • Reue K.
      • Lusis A.J.
      PON3 knockout mice are susceptible to obesity, gallstone formation, and atherosclerosis.
      ). ENaC and the bile acid–sensitive ion channel, a related member of the ENaC/degenerin family, are expressed in cholangiocytes lining bile ducts, and their channel activities can be regulated by unconjugated and conjugated bile acids (
      • Wiemuth D.
      • Sahin H.
      • Falkenburger B.H.
      • Lefèvre C.M.
      • Wasmuth H.E.
      • Gründer S.
      BASIC: a bile acid-sensitive ion channel highly expressed in bile ducts.
      ,
      • Wiemuth D.
      • Sahin H.
      • Lefèvre C.M.
      • Wasmuth H.E.
      • Gründer S.
      Strong activation of bile acid-sensitive ion channel (BASIC) by ursodeoxycholic acid.
      ,
      • Li Q.
      • Kresge C.
      • Bugde A.
      • Lamphere M.
      • Park J.Y.
      • Feranchak A.P.
      Regulation of mechanosensitive biliary epithelial transport by the epithelial Na+ channel.
      ,
      • Wang X.P.
      • Im S.J.
      • Balchak D.M.
      • Montalbetti N.
      • Carattino M.D.
      • Ray E.C.
      • Kashlan O.B.
      Murine epithelial sodium (Na+) channel regulation by biliary factors.
      ).
      As the least-studied member in the PON family, little is known about the expression or function of PON3 in other organs, such as the kidneys. We show, for the first time, that Pon3 is specifically expressed in distal nephron segments of mouse kidneys, including the DCT2, CNT, and CD. These are the same nephron segments where ENaC resides. In the CNT and CD, fine-tuning of Na+ reabsorption takes place under the control of aldosterone (
      • Loffing J.
      • Pietri L.
      • Aregger F.
      • Bloch-Faure M.
      • Ziegler U.
      • Meneton P.
      • Rossier B.C.
      • Kaissling B.
      Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and low-Na diets.
      ,
      • Mick V.E.
      • Itani O.A.
      • Loftus R.W.
      • Husted R.F.
      • Schmidt T.J.
      • Thomas C.P.
      The α-subunit of the epithelial sodium channel is an aldosterone-induced transcript in mammalian collecting ducts, and this transcriptional response is mediated via distinct cis-elements in the 5′-flanking region of the gene.
      ,
      • Loffing J.
      • Zecevic M.
      • Féraille E.
      • Kaissling B.
      • Asher C.
      • Rossier B.C.
      • Firestone G.L.
      • Pearce D.
      • Verrey F.
      Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK.
      ,
      • Náray-Fejes-Tóth A.
      • Fejes-Tóth G.
      The sgk, an aldosterone-induced gene in mineralocorticoid target cells, regulates the epithelial sodium channel.
      ,
      • Blazer-Yost B.L.
      • Liu X.
      • Helman S.I.
      Hormonal regulation of ENaCs: insulin and aldosterone.
      ,
      • Gründer S.
      • Rossier B.C.
      A reappraisal of aldosterone effects on the kidney: new insights provided by epithelial sodium channel cloning.
      ,
      • Denault D.L.
      • Fejes-Tóth G.
      • Náray-Fejes-Tóth A.
      Aldosterone regulation of sodium channel γ-subunit mRNA in cortical collecting duct cells.
      ,
      • Schafer J.A.
      • Hawk C.T.
      Regulation of Na+ channels in the cortical collecting duct by AVP and mineralocorticoids [editorial].
      ). Interestingly, we observed differential expression of Pon3 in distal nephron segments. It appears to have homogeneous expression within the NCC-positive DCT2 and CaBP28K-postive CNT, where Pon3 is nearly present in all cells lining these tubules. In contrast, Pon3 has higher expression in V-ATPase-positive ICs than in AQP2-positive PCs of CDs. This is consistent with a previous study showing that higher levels of Pon3 transcripts were found in ICs of mouse CDs (
      • Chen L.
      • Lee J.W.
      • Chou C.L.
      • Nair A.V.
      • Battistone M.A.
      • Păunescu T.G.
      • Merkulova M.
      • Breton S.
      • Verlander J.W.
      • Wall S.M.
      • Brown D.
      • Burg M.B.
      • Knepper M.A.
      Transcriptomes of major renal collecting duct cell types in mouse identified by single-cell RNA-seq.
      ). In addition, the expression of Pon3 is quite variable in PCs of CDs, with only ∼25% cells coexpressing both Pon3 and AQP2.
      This is particularly intriguing because the magnitude of ENaC-mediated Na+ reabsorption differs in these segments. It has been shown that CD-specific αENaC KO mice were able to maintain normal Na+/K+ homeostasis when challenged with dietary salt/water restriction (
      • Rubera I.
      • Loffing J.
      • Palmer L.G.
      • Frindt G.
      • Fowler-Jaeger N.
      • Sauter D.
      • Carroll T.
      • McMahon A.
      • Hummler E.
      • Rossier B.C.
      Collecting duct-specific gene inactivation of αENaC in the mouse kidney does not impair sodium and potassium balance.
      ). When αENaC was deleted in the CNT and CD, KO mice developed a severe salt-wasting phenotype and a continuous reduction in body weight (
      • Christensen B.M.
      • Perrier R.
      • Wang Q.
      • Zuber A.M.
      • Maillard M.
      • Mordasini D.
      • Malsure S.
      • Ronzaud C.
      • Stehle J.C.
      • Rossier B.C.
      • Hummler E.
      Sodium and potassium balance depends on αENaC expression in connecting tubule.
      ). In addition, ENaC currents are significantly greater in the CNT compared with the cortical CD (
      • Nesterov V.
      • Dahlmann A.
      • Krueger B.
      • Bertog M.
      • Loffing J.
      • Korbmacher C.
      Aldosterone-dependent and -independent regulation of the epithelial sodium channel (ENaC) in mouse distal nephron.
      ,
      • Frindt G.
      • Palmer L.G.
      Na channels in the rat connecting tubule.
      ). Together, these studies suggest that ENaC expression in the CNT has a key role in Na+ homeostasis in mice. As PON3 is mainly expressed in principal cells of the CNT, we predict that PON3 has an important role in regulating renal Na+/K+ homeostasis and BP control.
      This study examined the role of PON3 in regulating ENaC functional expression in different expression systems. We found that Pon3 KD in mCCD cells led to enhanced amiloride-sensitive Na+ transport, a higher abundance of a cleaved γENaC product (∼75 kDa), and higher ratios of cleaved γENaC versus total γENaC, suggesting up-regulation of ENaC proteolytic processing and surface expression in mCCD cells when Pon3 expression was reduced. Supporting this notion, surface expression of ENaC subunits was reduced by Pon3 coexpression in FRT cells and Xenopus oocytes. It is interesting to note that the anti-γENaC antibody recognized two forms (cleaved and uncleaved) of endogenous γENaC in mCCD cells but favored cleaved γENaC when ENaC was overexpressed in FRT cells. Nonetheless, our results using cultured epithelial cells or Xenopus oocytes support that PON3 decreases ENaC functional expression. Other members of this family, including PON2 and MEC-6, also inhibit ENaC activity in oocytes (
      • Shi S.
      • Buck T.M.
      • Kinlough C.L.
      • Marciszyn A.L.
      • Hughey R.P.
      • Chalfie M.
      • Brodsky J.L.
      • Kleyman T.R.
      Regulation of the epithelial Na+ channel by paraoxonase-2.
      ). This negative regulation of channel activity is of great interest to us, as ENaC hyperactivity has physiological consequences (
      • 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.
      ,
      • Shimkets R.A.
      • Warnock D.G.
      • Bositis C.M.
      • Nelson-Williams C.
      • Hansson J.H.
      • Schambelan M.
      • Gill Jr., J.R.
      • Ulick S.
      • Milora R.V.
      • Findling J.W.
      Liddle's syndrome: heritable human hypertension caused by mutations in the β subunit of the epithelial sodium channel.
      ,
      • 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.
      ,
      • Fang X.
      • Fukuda N.
      • Barbry P.
      • Sartori C.
      • Verkman A.S.
      • Matthay M.A.
      Novel role for CFTR in fluid absorption from the distal airspaces of the lung.
      ,
      • Mall M.A.
      • Button B.
      • Johannesson B.
      • Zhou Z.
      • Livraghi A.
      • Caldwell R.A.
      • Schubert S.C.
      • Schultz C.
      • O'Neal W.K.
      • Pradervand S.
      • Hummler E.
      • Rossier B.C.
      • Grubb B.R.
      • Boucher R.C.
      Airway surface liquid volume regulation determines different airway phenotypes in Liddle compared with βENaC-overexpressing mice.
      ,
      • Rauh R.
      • Diakov A.
      • Tzschoppe A.
      • Korbmacher J.
      • Azad A.K.
      • Cuppens H.
      • Cassiman J.J.
      • Dötsch J.
      • Sticht H.
      • Korbmacher C.
      A mutation of the epithelial sodium channel associated with atypical cystic fibrosis increases channel open probability and reduces Na+ self inhibition.
      ). There is growing evidence suggesting that members of the PON family, including MEC-6, PON2, and PON3, function as chaperones to modulate the expression of ion channels within the ENaC/degenerin family (
      • Chen Y.
      • Bharill S.
      • Altun Z.
      • O'Hagan R.
      • Coblitz B.
      • Isacoff E.Y.
      • Chalfie M.
      Caenorhabditis elegans paraoxonase-like proteins control the functional expression of DEG/ENaC mechanosensory proteins.
      ,
      • Shi S.
      • Buck T.M.
      • Kinlough C.L.
      • Marciszyn A.L.
      • Hughey R.P.
      • Chalfie M.
      • Brodsky J.L.
      • Kleyman T.R.
      Regulation of the epithelial Na+ channel by paraoxonase-2.
      ). MEC-6 is not only required for proper folding and assembly of MEC-4 channels but also to facilitate channel surface expression (
      • Chen Y.
      • Bharill S.
      • Altun Z.
      • O'Hagan R.
      • Coblitz B.
      • Isacoff E.Y.
      • Chalfie M.
      Caenorhabditis elegans paraoxonase-like proteins control the functional expression of DEG/ENaC mechanosensory proteins.
      ). Little is known regarding the chaperone function of PONs. We found that coexpression of Pon3 reduced ENaC whole-cell expression in FRT cells, suggesting that this trimeric channel is less stable in the presence of Pon3. Molecular chaperones have important roles in ENaC quality control within the early secretory pathway (
      • Buck T.M.
      • Kolb A.R.
      • Boyd C.R.
      • Kleyman T.R.
      • Brodsky J.L.
      The endoplasmic reticulum-associated degradation of the epithelial sodium channel requires a unique complement of molecular chaperones.
      ,
      • Buck T.M.
      • Plavchak L.
      • Roy A.
      • Donnelly B.F.
      • Kashlan O.B.
      • Kleyman T.R.
      • Subramanya A.R.
      • Brodsky J.L.
      The Lhs1/GRP170 chaperones facilitate the endoplasmic reticulum-associated degradation of the epithelial sodium channel.
      ,
      • You H.
      • Ge Y.
      • Zhang J.
      • Cao Y.
      • Xing J.
      • Su D.
      • Huang Y.
      • Li M.
      • Qu S.
      • Sun F.
      • Liang X.
      Derlin-1 promotes ubiquitylation and degradation of the epithelial Na+ channel, ENaC.
      ). It is possible that PON3 facilitates proteasomal degradation of misfolded ENaC subunits. Other chaperones have been identified that affect ENaC trafficking at later sites in the secretory pathway (
      • Kashlan O.B.
      • Mueller G.M.
      • Qamar M.Z.
      • Poland P.A.
      • Ahner A.
      • Rubenstein R.C.
      • Hughey R.P.
      • Brodsky J.L.
      • Kleyman T.R.
      Small heat shock protein αA-crystallin regulates epithelial sodium channel expression.
      ,
      • Chanoux R.A.
      • Robay A.
      • Shubin C.B.
      • Kebler C.
      • Suaud L.
      • Rubenstein R.C.
      Hsp70 promotes epithelial sodium channel functional expression by increasing its association with coat complex II and its exit from endoplasmic reticulum.
      ,
      • Chanoux R.A.
      • Shubin C.B.
      • Robay A.
      • Suaud L.
      • Rubenstein R.C.
      Hsc70 negatively regulates epithelial sodium channel trafficking at multiple sites in epithelial cells.
      ,
      • Grumbach Y.
      • Bikard Y.
      • Suaud L.
      • Chanoux R.A.
      • Rubenstein R.C.
      ERp29 regulates epithelial sodium channel functional expression by promoting channel cleavage.
      ,
      • Grifoni S.C.
      • McKey S.E.
      • Drummond H.A.
      Hsc70 regulates cell surface ASIC2 expression and vascular smooth muscle cell migration.
      ). Additional studies are needed to investigate mechanisms by which PONs regulate ENaC expression and the physiological relevance of this regulation in the kidneys and other organs.

      Experimental procedures

      Plasmids

      Mouse Pon3 was cloned in the pCMV6 vector (MR220409, OriGene) and has a C-terminal Myc-FLAG tag. The mouse ENaC α, β, and γ subunits with an N-terminal HA epitope tag and a C-terminal V5 epitope tag were cloned in the pcDNA3 vector and used to transfect FRT cells. WT or mutant mouse ENaC α, β, and γ subunits cloned in the pBlueScript vector were used for expressing ENaC in oocytes. Mouse γGT was cloned in the pcDNA3 vector (
      • Joyce-Brady M.
      • Jean J.C.
      • Hughey R.P.
      γ-Glutamyltransferase and its isoform mediate an endoplasmic reticulum stress response.
      ).

      Tissue immunofluorescence staining

      Kidneys from WT C57BL/6 mice or Pon3 KO mice (The Jackson Laboratory, 027311) were harvested using a protocol approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Formalin-fixed kidney tissue was embedded in paraffin and cut into 4-μm-thick serial sections. After deparaffinization and rehydration, the tissues were blocked with 10% horse serum to block nonspecific staining. Kidney sections were incubated with the following primary antibodies overnight at 4 °C: goat anti-PON3 (2 μg/ml, R&D Systems, AF4345), rabbit anti-Na–K–Cl cotransporter (1 μg/ml, StressMarq, SPC401), guinea pig anti-parvalbumin (2 μg/ml, Swant, GP72), rabbit anti-NCC (2 μg/ml, StressMarq, SPC401), rabbit anti-CaBP28K (0.5 μg/ml, Proteintech, 144791AP), rabbit anti-AQP2 (0.6 μg/ml, Alomone Labs, AQP-002), and rabbit anti-V-ATPase E1 subunit (1 μg/ml, Invitrogen, PA5-29899). The sections were subsequently incubated with fluorescent secondary donkey antibodies (anti-guinea pig Alexa 647 (2 μg/ml), anti-goat Alexa 488 (4 μg/ml), or anti-rabbit Cy3 (2 μg/ml), Jackson ImmunoResearch Laboratories) for 2 h at room temperature. A Leica SP8 confocal microscope was used to image transverse sectioned kidneys from the cortex to the medulla using a tile scan technique and a 40× oil, 1.3 numerical aperture objective. Other images were acquired with a Leica DM6000B wide-field microscope with a Retiga 4000R Fast 1394 camera. To quantify Pon3 expression in specific nephron segments and cell types, the staining was repeated with kidney tissues from four WT mice, and four or five fields were chosen randomly for each kidney sample. The fluorescence intensity of Pon3 staining in glomeruli was used as the threshold to identify positive Pon3 staining. Numbers of total cells as well as cells with positive staining of Pon3 and/or the chosen markers were manually counted for each individual PON3-positive tubule in the field. The specificity of the anti-PON3 antibody was examined in kidney sections from Pon3 KO mice.

      mCCD cells, siRNA KD, and Ussing chamber recording

      mCCD cells were grown in DMEM/F12 medium (Gibco, 21041) supplemented with insulin (5 μg/ml), human apotransferrin (5 μg/ml), epidermal growth factor (10 ng/ml), tri-iodothyronine (1 nm), dexamethasone (50 nm), sodium selenite (0.06 nm), and 2% decomplemented FBS at 37 °C in 5% CO2 as described previously (
      • Klemens C.A.
      • Edinger R.S.
      • Kightlinger L.
      • Liu X.
      • Butterworth M.B.
      Ankyrin G expression regulates apical delivery of the epithelial sodium channel (ENaC).
      ). For KD experiments, cells were reverse-transfected with 50 pmol of a control siRNA of scrambled sequence or a dicer-specific siRNA targeting the mouse Pon3 sequence 5′-GUACUAUAUUUCACAAAGCUCUGTA-3′ (Integrated DNA Technologies) on permeable Snapwell filters (1.12-cm2 surface area) using Lipofectamine 3000 (L3000008, Invitrogen) according to the manufacturer's instructions. Transfected cells were cultured for an additional 4 days to fully polarize before being mounted onto modified Ussing chambers (P2302, Physiological Instruments) for electrophysiology experiments.
      The hemichambers contained 5 ml of Krebs buffer solution (110 mm NaCl, 25 mm NaHCO3, 5.8 mm KCl, 2 mm MgSO4, 1.2 mMK2HPO4, 2 mm CaCl2, and 11 mm glucose) in both sides. The hemichambers were continuously bubbled with 95% O2, 5% CO2, which maintained the pH at 7.4 and the temperature at 37 °C. Isc was measured under voltage clamp conditions. After Isc reached a stable state, 10 μm amiloride was added to the apical side. To calculate the transepithelial resistance, a bipolar pulse of 10 mV with a duration of 0.5 s was applied every 60 s. All filters were recovered to collect whole-cell lysates with a detergent solution (20 mm HEPES, 100 mm NaCl, 40 mm KCl, 1 mm EDTA, 10% glycerol, 1% NP40, and 0.4% deoxycholate (pH 7.4)) supplemented with protease inhibitor mixture III (535140, Calbiochem). Whole-cell lysates were subjected to SDS-PAGE under reducing conditions to probe for the endogenous Pon3 (0.1 μg/ml, Sigma, HPA014848), γENaC (1 μg/ml, StressMarq, SPC-405), or actin (0.2 μg/ml, Sigma, A1978). Immunoblots were developed using Clarity Western ECL blotting substrate (1705060, Bio-Rad) and imaged with Bio-Rad ChemiDocTM.

      FRT cell coimmunoprecipitation and surface biotinylation

      FRT cells were cultured in DMEM/F12 medium supplemented with 8% FBS. For the coimmunoprecipitation assay, FRT cells were seeded on 6-well dishes and transfected with plasmids encoding mouse Pon3 and mouse ENaC subunits (0.5 μg/construct). In each case, only one ENaC subunit had an N-terminal HA tag and a C-terminal V5 epitope tag (HAαV5βγ, αHAβV5γ or αβHAγV5). The next day, cells were extracted with detergent solution supplemented with protease inhibitor mixture. Five percent of the cell lysate was saved as input. The remainder was incubated overnight with 50 μl of agarose-immobilized anti-V5 antibodies (S190-119, Bethyl) with end-over-end mixing at 4 °C to pull down ENaC subunits. To pull down Pon3, the lysates were incubated with anti-FLAG antibodies (2 μg, F3165, Sigma) in the presence of rec-protein G–Sepharose (10-1241, Invitrogen). Proteins were eluted into Laemmli sample buffer (1610737, Bio-Rad) by heating the isolated beads at 95 °C. The immunoprecipitate and the input were subjected to SDS-PAGE and blotted for Pon3 (0.1 μg/ml, Sigma, HPA014848) or for ENaC subunits with V5 antibody (0.2 μg/ml, Invitrogen, R96025). Assays were repeated three times for each condition.
      To measure surface expression of ENaC, FRT cells were transfected with plasmids encoding mouse HAαV5βγ with or without the Pon3 plasmid (0.5 μg/construct) directly on 24-mm Transwell filters and cultured to confluency. In the control group, an equal amount of the pCMV6 vector was included. FRT cells grown on the filters were washed four times with cold Dulbecco's PBS with 1.0 mm CaCl2 and 0.5 mm MgCl2 (PBS, Corning Life Sciences). To label apical surface proteins, 1 mg/ml EZ-LinkTM Sulfo-NHS-SS-Biotin (21331, Thermo Fisher) in a buffer containing 137 mm NaCl and 15 mm sodium borate (pH 9.0) was added to the apical side, whereas the basolateral side was incubated with just the buffer. Excess biotin was then quenched with 10% FBS in DMEM/F12. After washing with PBS, cells were lysed in detergent solution and incubated with NeutrAvidin agarose (29200, Thermo Fisher) overnight at 4 °C to isolate biotinylated proteins. The recovered surface proteins and 5% of the total lysate were separated by SDS-PAGE and blotted for the α subunit with HA-HRP antibodies (0.05 μg/ml, 3F10, Sigma), the γ subunit (0.3 μg/ml, StressMarq), PON3 (0.03 μg/ml, Sigma), or GAPDH (0.3 μg/ml, Proteintech, HRP-60004), as described previously (
      • Heidrich E.
      • Carattino M.D.
      • Hughey R.P.
      • Pilewski J.M.
      • Kleyman T.R.
      • Myerburg M.M.
      Intracellular Na+ regulates epithelial Na+ channel maturation.
      ,
      • Kashlan O.B.
      • Kinlough C.L.
      • Myerburg M.M.
      • Shi S.
      • Chen J.
      • Blobner B.M.
      • Buck T.M.
      • Brodsky J.L.
      • Hughey R.P.
      • Kleyman T.R.
      N-linked glycans are required on epithelial Na+ channel subunits for maturation and surface expression.
      ).
      In some experiments, FRT cells seeded on 6-well size plates were transfected with plasmids encoding mouse ENaC (HAαV5βγ) or mouse γGT (0.5 μg/construct). An equal amount of the pCDNA3 vector was used in the control (−γGT) group. The next day, whole-cell lysates were extracted with detergent solution supplemented with protease inhibitors and subjected to SDS-PAGE. αENaC was detected with HA-HRP antibodies (0.05 μg/ml, 3F10, Sigma). γGT was detected using a rabbit polyclonal antibody described previously (
      • Hughey R.P.
      • Altman R.A.
      • Wells W.J.
      • Curto K.A.
      Evidence for stable homodimers and heterodimers of γ-glutamyltranspeptidase subunits under protein-denaturing conditions.
      ).

      Xenopus oocyte expression, two-electrode voltage clamp, and surface expression

      cRNAs of mouse ENaC α, β, and γ subunits; mouse Pon3; and rat Romk were synthesized using mMESSAGE mMACHINETM transcription kits (Invitrogen) following the manufacturer's protocol. Oocytes were harvested from Xenopus laevis following a protocol approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Stage V-VI oocytes were injected with 2 ng of cRNA per ENaC subunit or 1 ng of rat Romk cRNA. An equal amount of mouse Pon3 cRNA was injected for the coexpression assay. The injected oocytes were incubated in modified Barth's saline (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 15 mm HEPES, 0.3 mm Ca(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4, 10 μg/ml sodium penicillin, 10 μg/ml streptomycin sulfate, and 100 μg/ml gentamycin sulfate (pH 7.4)) at 18 °C for an additional 24–48 h for optimal channel expression. Whole-cell Na+ currents were measured by clamping oocytes at −100 mV, whereas oocytes were continuously perfused with a solution containing 110 mm NaCl, 2 mm KCl, 1.6 mm CaCl2, and 10 mm HEPES (pH adjusted to 7.4). To examine the effect of Pon3 on ENaC gating in response to MTSET, oocytes were injected with cRNAs encoding the WT mouse ENaC α and γ subunits and the degenerin mutation βS518C with or without an equal amount of mouse Pon3 cRNA. MTSET was freshly prepared and delivered to oocytes through perfusion. To measure the Pon3 effect on Romk activity, we replaced the NaCl in the perfusion solution with 100 mm KCl. At the end of each recording, BaCl2 was applied at a final concentration of 5 mm to block Romk activity. Voltage clamping was performed using a GeneClamp 500B amplifier and DigiData 1440A interface (Molecular Devices). Electrophysiological data were analyzed with Clampfit 10.5 and plotted with Origin 2015 (OriginLab).
      ENaC surface expression in oocytes was measured using a chemiluminescence assay as described previously (
      • Carattino M.D.
      • Hill W.G.
      • Kleyman T.R.
      Arachidonic acid regulates surface expression of epithelial sodium channels.
      ). The mouse ENaC β subunit with an extracellular FLAG epitope tag was included in the cRNA mixture. A nontagged WT β subunit was used as a negative control. Surface expression was assessed 2 days after cRNA injection by incubating oocytes with anti-FLAG M2 antibodies (1 μg/ml, Sigma F3165) and then with anti-mouse HRP antibodies (1 μg/ml, Jackson ImmunoResearch Laboratories, 115036072). Chemiluminescence of each oocyte was developed with Super Signal ELISA Femto Maximum Sensitivity Substrate (37075, Thermo Scientific) and quantified as relative light units with a GloMax-Multi detection system (Promega). To investigate the effect of Pon3 on ENaC whole-cell expression, oocytes injected with ENaC or ENaC and Pon3 were homogenized, and whole-cell lysates were subjected to SDS-PAGE to probe for βENaC (1 μg/ml, StressMarq, SPC-404D) or GAPDH (0.3 μg/ml). All oocytes experiments were repeated at least three times using different frogs.

      Statistical analyses

      Data were expressed as the mean ± S.D. in the main text and are shown as scatter-dot plots, with a horizontal bar indicating the mean. Data distribution was examined with a D'Agostino–Pearson normality test. Statistical comparisons between two groups were determined with a nonparametric Mann–Whitney test. Statistical comparisons between three or more groups were performed with one-way or two-way ANOVA, followed by Dunn's or Sidak's multiple comparisons test, using Prism 8 (GraphPad Software, San Diego, CA). p < 0.05 was considered statistically significant

      Author contributions

      S. S. and T. R. K. conceptualization; S. S., N. M., X. W., B. M. R., A. L. M., and C. J. B. data curation; S. S., N. M., and X. W. formal analysis; S. S. and T. R. K. supervision; S. S., C. J. B., and T. R. K. funding acquisition; S. S. validation; S. S. investigation; S. S., C. J. B., and R. J. T. visualization; S. S. writing-original draft; S. S. and T. R. K. project administration; S. S., N. M., C. J. B., R. J. T., M. D. C., and T. R. K. writing-review and editing; B. M. R., A. L. M., C. J. B., R. J. T., and M. D. C. methodology; M. D. C. resources.

      Acknowledgments

      The γGT plasmid and the rabbit anti-γGT antibody were generous gifts from Dr. Rebecca P. Hughey from the Renal-Electrolyte Division at the University of Pittsburgh. We thank Dr. Hughey and Carol L. Kinlough for technical assistance with biochemical studies.

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