Project Abstract The epithelial sodium channel (ENaC) mediates the rate-limiting step of Na+ uptake across the apical membrane of specific epithelia. ENaC-dependent Na+ absorption in the kidney has important roles in regulating extracellular fluid volume, blood pressure and extracellular [K+]. Functional ENaC complexes in the kidney consist of three homologous subunits, namely α, β, and γ. ENaC functional expression is tightly regulated by multiple intracellular and exogenous factors. Several molecular chaperones have been implicated in key steps during ENaC biogenesis. The major goal of this proposal is to investigate the mechanism by which PON-2 regulates ENaC functional expression. PON-2 is a membrane-bound protein that shares structural features with MEC-6, a ER-resident chaperone of C. elegans. MEC-6 is required for the proper assembly and surface expression of the touch-sensing MEC-4/MEC-10 channel in worm touch-receptor neurons. We recently reported that PON-2 is expressed in principal cells of the distal nephron, where ENaC resides. PON-2 co-immunoprecipitates with ENaC subunits when co-expressed in HEK293 cells, and reduces ENaC activity when co-expressed in Xenopus oocytes. The PON-2– dependent reduction of ENaC activity is associated with a reduction in the number of Na+ channels at the cell surface. PON-2 lactonase activity is not required for its inhibitory effect on ENaC activity, suggesting that PON-2, like MEC-6, may function as a molecular chaperone to regulate ENaC expression. In this application, we propose to examine whether PON-2 reduces ENaC surface expression by impeding ENaC biogenesis and/or its forwarding trafficking, or by accelerating ENaC degradation via proteasome or lysosome pathways, by pharmacologically blocking specific steps of ENaC turnover or by employing mutant αβγ channels that are defective in gating or endocytosis (Liddle mutations). Another goal is to determine the physiological role of PON-2 in ENaC expression in Fisher rat thyroid (FRT) cell monolayers. We will examine whether ENaC expression and/or channel activity in FRT cells is altered under conditions in which PON- 2 is either overexpressed or knocked out. As a proof of concept, we propose to generate a Pon-2 knockout FRT cell line with CRISPR/Cas9 technology to study the physiological role of endogenous PON-2 in ENaC expression. These studies will provide key preliminary data for future studies of ENaC regulation by PON-2 in murine Pon-2 knockout models. Understanding mechanisms by which PON-2 regulates ENaC expression will provide novel insights into both normal and altered ENaC functional states, such as hypertension.