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

Shujie Shi,Nicolas Montalbetti,Xueqi Wang,Brittney M Rush,Allison L Marciszyn,Catherine J Baty,Roderick J Tan,Marcelo D Carattino,Thomas R Kleyman

doi:10.1074/jbc.ra119.011789

Abstract

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.

Highlights

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
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

Summary

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The canonical ENaC consists of three homologous subunits: ␣, ␤, and ␥ (25, 26). The fourth ENaC subunit, ␦ has been identified in multiple tissues of primates and other species (27, 28). All three PONs have antioxidative and anti-atherosclerotic properties (46 –50), they differ in their tissues expression and substrate olar Hϩ-ATPase; Isc, short circuit current; NC, negative control; ␥GT, ␥-glutamyl transferase; ROMK, renal outer medullary Kϩ channel; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate; ANOVA, analysis of variance; mCCD, mouse cortical collecting duct; PO, open probability. We have shown recently that PON2 inhibits ENaC activity by reducing channel surface expression (57), suggesting that the chaperone function is conserved between mammalian PONs and their nematode orthologs. This study examined the expression of Pon[3] in the mouse kidney and its effect on ENaC functional expression by silencing endogenous Pon[3] in mouse cortical collecting duct cells or overexpressing Pon[3] in cultured FRT cells or Xenopus oocytes

Results

Discussion

Experimental procedures