Regulation of epithelial sodium channel trafficking by proprotein convertase subtilisin/kexin type 9 (PCSK9)

Abstract

The epithelial Na(+) channel (ENaC) is critical for Na(+) homeostasis and blood pressure control. Defects in its regulation cause inherited forms of hypertension and hypotension. Previous work found that ENaC gating is regulated by proteases through cleavage of the extracellular domains of the α and γ subunits. Here we tested the hypothesis that ENaC is regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), a protease that modulates the risk of cardiovascular disease. PCSK9 reduced ENaC current in Xenopus oocytes and in epithelia. This occurred through a decrease in ENaC protein at the cell surface and in the total cellular pool, an effect that did not require the catalytic activity of PCSK9. PCSK9 interacted with all three ENaC subunits and decreased their trafficking to the cell surface by increasing proteasomal degradation. In contrast to its previously reported effects on the LDL receptor, PCSK9 did not alter ENaC endocytosis or degradation of the pool of ENaC at the cell surface. These results support a role for PCSK9 in the regulation of ENaC trafficking in the biosynthetic pathway, likely by increasing endoplasmic reticulum-associated degradation. By reducing ENaC channel number, PCSK9 could modulate epithelial Na(+) absorption, a major contributor to blood pressure control.

FIGURE 1.

PCSK9 inhibits ENaC current. A and B, Xenopus oocytes were nuclear-injected with cDNAs encoding human α-, β-, and γENaC (0.2 ng each) and PCSK9 (0–2 ng). A, representative current traces (0.2 ng PCSK9). 10 μm amiloride (Amil) was added to the bathing solution as indicated by the black bar. B, summary plot of amiloride-sensitive current versus amount of injected PCSK9 cDNA (mean ± S.E. relative to 0 PCSK9 group, n = 5–26). C and D, FRT epithelia were transfected with α-, β-, and γENaC (0.03 μg each) and PCSK9 (0–0.9 μg). C, representative short-circuit current traces. 10 μm amiloride was added to the apical bathing solution as indicated by the black bar. D, summary plot of amiloride-sensitive current versus the amount of transfected PCSK9 (mean ± S.E. relative to 0 PCSK9 group, n = 9–12). E and F, Xenopus oocytes were nuclear-injected with cDNAs encoding human ASIC1 (0.6 ng) and PCSK9 or control plasmid (0.8 ng). E, representative current traces. The bath was perfused with pH 5 solution as indicated by the black bar. F, summary plot of proton-activated current (mean ± S.E.; n = 9; *, p < 0.03).

FIGURE 2.

PCSK9 interacts with ENaC. A, coimmunoprecipitation of ENaC and PCSK9 in HEK 293 cells transfected with α-, β-, and γENaC (1 μg each) with or without PCSK9-V5 (3 μg). One of the ENaC subunits contained a FLAG epitope (coexpressed with the other two untagged ENaC subunits). Total cDNA 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 cell lysates for ENaC and PCSK9, as indicated. Full-length and cleaved forms of PCSK9 and ENaC are indicated. The data are representative of three experiments. B, coimmunoprecipitation in HEK 293 cells transfected with a single ENaC subunit (αENaC-FLAG, βENaC-FLAG, or γENaC-FLAG, 1 μg) with or without PCSK9-V5 (1 μg). The proteins were immunoprecipitated and immunoblotted as in A. The data are representative of three experiments.

FIGURE 3.

PCSK9 reduces ENaC cell surface expression. A, immunoblot analyses of ENaC in the cell surface biotinylated fraction (top panel) and in the total cell lysate (bottom panel) from HEK 293 cells transfected with α-, β-, and γENaC (1 μg each, one subunit contained FLAG epitope) with or without PCSK9 (3 μg). Total cDNA was kept constant using GFP cDNA. ENaC protein in +PCSK9 group relative to -PCSK9 group is quantified by densitometry in B (mean ± S.E.; n = 3–5; *, p < 0.03). C, immunoblots of Nedd4-2-HA (anti-HA) and β-actin in HEK 293 cells transfected with Nedd4-2-HA (3 μg) with or without PCSK9 (3 μg). D, amiloride-sensitive current in Xenopus oocytes expressing human α- and γENaC with wild-type or mutant βENaC (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 α_Y644AENaC-FLAG coexpressed with β_Y620A- and γ_Y627AENaC (1 μg each) with PCSK9 or GFP (3 μg). F, immunoblot of biotinylated (top panel) and total (bottom panel) αENaC-FLAG coexpressed in HEK 293 cells with β- and γENaC (1 μg each) with GFP or PCSK9 (wild type or S386A, 3 μg). Irrelevant lanes were removed digitally.

FIGURE 4.

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

FIGURE 5.

PCSK9 decreases ENaC exocytosis. Short-circuit currents were recorded in FRT epithelia transfected with α-, β-, and γ_G536CENaC subunits (0.16 μg each) with or without PCSK9 (0.5 μg). 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 of the 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 α_Cl-2 ENaC-FLAG in HEK 293 cells cotransfected with β- and γENaC (1 μg each) with or without PCSK9 (3 μg). The cells were treated with 5 μg/ml trypsin for 5 min, incubated at 37 °C for 0–60 min, and then biotinylated. B, quantification of the cleaved αENaC band relative to 0 min (mean ± S.E., n = 5).

FIGURE 7.

PCSK9 does not alter degradation of cell surface ENaC. A, immunoblots (anti-FLAG) of biotinylated αENaC in HEK 293 cells transfected with αENaC-FLAG, βENaC, and γENaC (1 μg each) with or without PCSK9 (3 μg). Cell surface proteins were pulse-labeled with biotin and then the cells were incubated at 37 °C for 0–120 min. B, biotinylated αENaC at each time was quantified relative to 0 min (mean ± S.E.; n = 3; *, p < 0.05).