Proteolytic activation of the epithelial sodium channel: role of pro‐protein convertases and prostasin

Abstract

The epithelial sodium channel (ENaC) is essential for regulating renal sodium excretion. Cleavage of the γENaC subunit increases ENaC activity both in vitro and in vivo; however, the involved endogenous proteases remain ambiguous. This study investigates the mechanistic roles of the pro‐protein convertase (PCSK) family and the serine protease prostasin in proteolytic activation of ENaC.We used the M‐1 mouse collecting duct cell line that natively expresses functional ENaC channels for pharmacological inhibition studies with pro‐protein convertase inhibitor decanoyl‐RVKR‐chloromethyl ketone (dec‐RVKR‐cmk) and serine protease inhibitor camostat mesilate (CM). Knockout cell lines of prostasin and three PCSKs were generated with the CRISPR/Cas9 system in M‐1 cells. As an index of ENaC activity, amiloride‐sensitive short‐circuit current (ISC) and transepithelial voltage (VTE) was measured on polarized cells on transwell filter supports in Ussing chambers. γENaC membrane levels and cleavage were tested by western blotting of surface biotinylated M‐1 cells with an antibody against the C‐terminal of γENaC. All values are means SE, compared with students t‐test or ANOVA as appropriate.Pro‐protein convertase inhibition decreased the amiloride‐sensitive ISC in M‐1 cells compared with controls (25.9 1.9 μA, n=5, vs. 45.7 7.0 μA, n=6, p<0.05). Similarly, serine protease inhibition decreased VTE (−34.5 7.8 mV, n=3, vs. −74.2 0.9 mV, n=3, p<0.05) but ISC was not significantly lower (68.0 8.5, n=3, vs. 74.0 2.3, n=3, n.s.). Preliminary evidence suggests that although both inhibitors decreased membrane expression of γENaC, only serine protease inhibitor treatment reduced γENaC cleavage (n=1). Knockout of the Psck3 (furin) gene by CRISPR completely abolished ENaC activity, while knockout of Pcsk4, Pcsk7, and prostasin all showed decreased ENaC activity compared to wild‐type M‐1 cells. Knockout of PCSKs reduced γENaC surface expression, but only knockout of prostasin reduced γENaC cleavage.We propose that PCSKs are not directly involved in proteolytic ENaC activation. Both pharmacological inhibition and knockout of prostasin decreased ENaC activity and γENaC cleavage, thus suggesting a potential role in proteolytic ENaC activation.Support or Funding Information The Danish Medical Research Council