Acetylation stimulates the epithelial sodium channel by reducing its ubiquitination and degradation

Abstract

The epithelial Na(+) channel (ENaC) functions as a pathway for Na(+) absorption in the kidney and lung, where it is crucial for Na(+) homeostasis and blood pressure regulation. ENaC is regulated in part through signaling pathways that control the ubiquitination state of ENaC lysines. A defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. Here we determined that α-, β-, and γENaC are also substrates for lysine acetylation. Trichostatin A (TSA), a histone deacetylase inhibitor, enhanced ENaC acetylation and increased ENaC abundance in the total cell lysate and at the cell surface. Moreover, TSA increased ENaC current in Fischer rat thyroid and kidney collecting duct epithelia. We found that HDAC7 is expressed in the kidney collecting duct, supporting a potential role for this histone deacetylase in ENaC regulation. HDAC7 overexpression reduced ENaC abundance and ENaC current, whereas ENaC abundance and current were increased by silencing of HDAC7. ENaC and HDAC7 form a complex, as detected by coimmunoprecipitation. We observed a reciprocal relationship between acetylation and ubiquitination; TSA reduced ENaC ubiquitination, whereas HDAC7 increased ubiquitination. By reducing ENaC ubiquitination, TSA decreased the rate of ENaC degradation. Thus, acetylation increases epithelial Na(+) absorption by antagonizing ENaC ubiquitination. This stabilizes ENaC, and hence, increases its abundance at the cell surface.

Keywords: Amiloride; Epithelia; Epithelial Sodium Channel (ENaC); Histone Deacetylase (HDAC); Hypertension; Lysine Acetylation; Protein Degradation; Ubiquitylation (Ubiquitination).

FIGURE 1.

ENaC acetylation. HEK 293 cells were transfected with cDNAs encoding α-FLAG, β-FLAG, or γ-FLAG (wild type or K-R) ENaC or empty plasmid (C) (2 μg), as indicated, and treated with TSA (700 nm) or vehicle (ethanol) for 24 h. Acetylated ENaC (ENaC-Ac) subunits were immunoprecipitated with anti-acetyl lysine antibody and immunoblotted with anti-FLAG antibody.

FIGURE 2.

Acetylation increases ENaC abundance. A, immunoblots (IB) (anti-FLAG) of total (top panel) and biotinylated (middle panel) ENaC subunits in HEK 293 cells cotransfected with α-, β-, and γENaC (2 μg of each, one subunit contained FLAG epitope (FL)) and treated with TSA (700 nm) or vehicle (ethanol) for 24 h. Immunoblots for β-actin are shown in the bottom panel. B, immunoblots (anti-FLAG) of total (top panel) and biotinylated (middle panel) αENaC in HEK 293 cells transfected with α_FLENaC (2 μg) and treated with TSA (700 nm) or vehicle (ethanol) for 24 h. Immunoblot for β-actin is shown in the bottom panel.

FIGURE 3.

Acetylation increases ENaC current. A, superimposed representative short-circuit current traces in FRT epithelia transfected with α-, β-, and γENaC (0.5 μg of each) and treated with TSA (50 nm, black) or vehicle (ethanol, gray) for 24 h. Amiloride (10 μm) was added to the apical bathing solution, as indicated by the black bar. B, quantification of amiloride-sensitive short-circuit currents (relative to −TSA groups) in FRT epithelia transfected with wild type ENaC or cytoplasmic K-R mutant ENaC (K-R) (mean ± S.E., n = 12–14; *, p < 0.0001). Non-normalized currents in the absence of TSA were: wild type, 2.82 ± 0.2 μA/cm²; K-R, 0.73 ± 0.03 μA/cm². C, representative short-circuit current traces in mpkCCD epithelia treated with TSA (50 nm, black) or vehicle (ethanol, gray) for 24 h. D, quantification of amiloride-sensitive short-circuit currents in mpkCCD epithelia (relative to −TSA group) (mean ± S.E., n = 14; *, p < 0.009).

FIGURE 4.

HDAC7 reduces ENaC abundance. A, HDAC7 immunoblots in kidney lysates from rat and mouse (Mse) and isolated glomeruli (Glom). B, immunohistochemistry of rat kidney sections using HDAC7 antibody (top panel) or aquaporin 2 (AQP2) antibody (bottom panel). Scale bars are 50 μm. C, immunoblots (IB) (anti-FLAG) of total (top panel) and biotinylated (middle panel) αENaC in HEK 293 cells cotransfected with α_FL-, β-, and γENaC (1 μg of each) with or without HDAC7 or HDAC9 (3 μg). Immunoblots for HDAC7 or HDAC9 are shown in the bottom panels. D, quantification of αENaC in total cell lysate in cells cotransfected with or without HDAC7 or HDAC9 (relative to −HDAC group) (mean ± S.E., n = 5; *, p < 0.005).

FIGURE 5.

HDAC7 reduces ENaC current. A, superimposed representative short-circuit current traces in FRT epithelia cotransfected with α-, β-, and γENaC (0.25 μg of each) and HDAC7 or GFP (−HDAC7)(0.75 μg). Amiloride (10 μm) was added to the bathing solution, as indicated by the black bar. B, quantification of amiloride-sensitive short-circuit currents in FRT epithelia (relative to −HDAC7 group) (mean ± S.E., n = 11; *, p < 0.01). C, immunoblot (IB) (anti-FLAG) of HDAC7-FLAG in mpkCCD epithelia transfected with HDAC7-FLAG (+) or GFP (−)(2.8 μg). D, quantification of amiloride-sensitive short-circuit currents in mpkCCD epithelia transfected with HDAC7 or GFP (relative to −HDAC7 group) (mean ± S.E., n = 15–16; *, p < 0.015). E, immunoblot of endogenous HDAC7 and αENaC in mpkCCD epithelia transfected with HDAC7 siRNA or non-targeting control (Cntrl) siRNA. F, quantification of amiloride-sensitive short-circuit currents in mpkCCD epithelia transfected with two HDAC7 siRNAs or control siRNA (relative to control siRNA group) (mean ± S.E., n = 14–22; *, p < 0.009).

FIGURE 6.

HDAC7 and αENaC form a complex. Coimmunoprecipitation of αENaC-V5 and HDAC7-HA in HEK 293 cells transfected with cDNAs encoding αENaC-V5, HDAC7-HA, or both was performed. Cell lysates were immunoprecipitated (IP) and immunoblotted (IB) as indicated.

FIGURE 7.

Acetylation antagonizes ENaC ubiquitination. A and C, immunoblots (IB) of ubiquitinated αENaC (αENaC-Ub) in total cell lysate (top panels) (immunoprecipitation of α_FL; immunoblotting of ubiquitin-HA and biotinylated cell surface fraction (middle panels) (immunoprecipitation of α_FL, followed by pulldown of biotinylated proteins with immobilized NeutrAvidin, and then immunoblotting of ubiquitin-HA), and total αENaC in the cell lysates (bottom panels). HEK 293 cells were transfected with α_FL-, β-, and γENaC (1 μg of each) and ubiquitin-HA (3 μg) and treated with TSA (700 nm for 24 h, +) or vehicle (ethanol, −) (A), or cotransfected with HDAC7 (+) or GFP (−) (C). B and D, the quantity of ubiquitinated αENaC (relative to −TSA (B) or −HDAC7 (D)) is plotted (mean ± S.E., n = 4–6; *, p < 0.007). In panel C, we used shorter exposures than in panel A to allow us to detect increased ubiquitination induced by HDAC7.

FIGURE 8.

Acetylation reduces ENaC degradation. A, immunoblots of αENaC-FLAG in HEK 293 cells cotransfected with β- and γENaC and treated with TSA (700 nm) or vehicle for 24 h. To inhibit protein synthesis, the cells were treated with cycloheximide (10 μg/ml) for 0–3 h prior to lysis. B, quantification of αENaC protein relative to 0 min time point (mean ± S.E.; n = 5–7).