Hsc70 negatively regulates epithelial sodium channel trafficking at multiple sites in epithelial cells

Abstract

The epithelial sodium channel (ENaC) plays an important role in homeostasis of blood pressure and of the airway surface liquid, and excess function of ENaC results in refractory hypertension (in Liddle's syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsc70, is not completely understood. Our previously published data suggest that Hsc70 negatively affects ENaC activity and surface expression in Xenopus oocytes; here we investigate the mechanism by which Hsc70 acts on ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsc70, treatment with 5 μg/ml doxycycline increased total Hsc70 expression 20%. This increase in Hsc70 expression led to a decrease in ENaC activity and surface expression that corresponded to an increased rate of functional ENaC retrieval from the cell surface. In addition, Hsc70 overexpression decreased the association of newly synthesized ENaC subunits. These data support the hypothesis that Hsc70 inhibits ENaC functional expression at the apical surface of epithelia by regulating ENaC biogenesis and ENaC trafficking at the cell surface.

Keywords: ENaC; chaperone; epithelia; heat shock protein; trafficking.

Fig. 1.

Hsc70 decreases epithelial sodium channel (ENaC) functional expression in Madin-Darby canine kidney (MDCK) cells. MDCK cells were grown as epithelial monolayers on semipermeable supports and incubated with doxycycline (Dox, 0–5 μg/ml) for 24 h, and amiloride-sensitive short-circuit current (I_sc) was determined in Ussing chambers. Hsc70-expressing (A) or ATPase-deficient Hsc70-expressing (B) cells were analyzed, and amiloride-sensitive I_sc data are expressed relative to samples incubated without Dox. Values are means ± SE for number of replicates shown above data points. *P < 0.05 vs. 0 Dox (by ANOVA).

Fig. 2.

Increased expression of Hsc70 in MDCK cell lysates. A: MDCK cells were treated with 5 μg/ml Dox for 0–24 h, and Hsc70 was detected in cell lysates by immunoblot (IB). Lower and upper bands represent endogenous Hsc70 protein and Myc/His-tagged construct of Hsc70 protein, respectively. B and C: immunoblot determination of Hsc70 and ATPase-deficient Hsc70 expression after 24 h of incubation with the indicated concentration of Dox. This immunoreactivity was compared with that of purified Hsc70 protein to quantify Hsc70 expression. The 0 μg/ml and 5 μg/ml and protein standard lanes in B are from noncontiguous portions of the same immunoblot. Densitometric analysis of 4 independent experiments quantifying endogenous (light bars) and overexpressed (dark bars) chaperone are shown. Error bars, SE.

Fig. 3.

Effect of increased Hsc70 expression on whole cell ENaC and Hsp70 expression in MDCK cells. MDCK cells were treated with Dox (0–5 μg/ml) for 24 h, and expression of the ENaC subunits was determined by immunoblot analysis using antibodies directed to the epitope tags on each of the subunits. Hsp70 immunoreactivity was similarly determined, and GAPDH immunoreactivity was used as a loading control. Immunoblots are representative of 3 independent experiments.

Fig. 4.

Hsc70 decreases surface expression of ENaC in MDCK cells. MDCK cells overexpressing Hsc70 were treated with 0 or 5 μg/ml Dox for 24 h. β-ENaC (A) or γ-ENaC (B) at the apical surface was detected by surface biotinylation. Representative immunoblots for β-ENaC (using an antibody to the V5 epitope tag, A) of whole cell lysates and NeutrAvidin-precipitated proteins are shown. Immunoblots for GAPDH were used as a control for protein loading and to ensure that the membrane-impermeant biotin did not label intracellular proteins. These panels are from noncontiguous lanes of the same immunoblot. Densitometric quantification of immunoblots from 3–4 independent biotinylation experiments is also shown. Error bars, SE. *P = 0.029 (n = 4 independent experiments; A) and 0.005 (n = 3 independent experiments; B) vs. 0 μg/ml Dox (by t-test).

Fig. 5.

Influence of Hsc70 on rate of ENaC retrieval from the apical cell surface. MDCK cells were treated without or with Dox for 24 h, and a baseline I_sc was established in Ussing chambers. Cycloheximide (100 μg/ml) was added, and amiloride-sensitive I_sc was determined at the indicated times. Values are means ± SE for n repeats. Pseudo-1st-order rate constants (k_app) for decay of amiloride-sensitive I_sc were determined by fitting these data to the following equation: (I_t/I₀) = e^−kt, where (I_t/I₀) is the fraction of ENaC current remaining at time t and k (k_app) is the apparent 1st-order rate constant, by a nonlinear regression using the Regression Wizard function of Sigma Plot (version 8.0). This analysis yields k_app ± SE, which were then compared by t-test as a function of ± Dox. A: cells expressing Hsc70. B: cells expressing ATPase-deficient Hsc70.

Fig. 6.

Increased Hsc70 expression increases relative expression of uncleaved vs. cleaved ENaC at the apical surface of MDCK cells. MDCK cells expressing Hsc70 were treated without or with Dox for 24 h, and a baseline I_sc was established in Ussing chambers. Trypsin (10 μg/ml) was applied to the apical surface to cleave and activate uncleaved/inactive ENaC channels, and amiloride-sensitive I_sc was determined. Left: amiloride-sensitive I_sc before (○) and after (▽) treatment with trypsin. Right: fraction of total amiloride-sensitive I_sc stimulated by trypsin, where data are presented as change in I_sc with trypsin relative to total amiloride-sensitive I_sc. Values are means ± SE for number of repeats indicated above each data point. *P < 0.05 vs. 0 μg/ml Dox control by 1-way ANOVA.

Fig. 7.

Overexpression of Hsc70 modulates colocalization between α-ENaC and Rab proteins. A: immunofluorescence detection of Rab7 (red) and α-ENaC (via HA tag, green) and their colocalization (yellow in merge image). Representative images of 4 independent experiments are shown. B: immunofluorescence detection of Rab11 (red) and α-ENaC (via HA tag, green) and their colocalization (yellow in merge image). Representative images of 4 independent experiments are shown.

Fig. 8.

Hsc70 influences association of ENaC with endocytic vesicle components. Cells were treated with 0 or 5 μg/ml Dox for 24 h to induce increased Hsc70 expression. Samples underwent immunoprecipitation with anti-Rab7 (A) or anti-Rab11 (B), and β-ENaC was detected in precipitated proteins by immunoblot using anti-V5. Input samples represent 10% of total protein subject to immunoprecipitation. Fold change in V5-β-ENaC coprecipitated with Rab7 or Rab11 was determined by densitometric analysis of precipitated V5 from 4 independent experiments. Values (means ± SE) are presented relative to control (0 μg/ml Dox). *P = 0.029 (A) and 0.018 (B) vs. 0 μg/ml Dox (by t-test). Immunoblots of total Rab7 (A) or Rab11 (B) present in whole cell lysates are included for samples treated with or without Dox and demonstrate that Dox did not alter expression of these species.

Fig. 9.

ATPase activity of Hsc70 is required for the effect of Hsc70 on ENaC association with endocytic vesicle components. MDCK cells were treated with 0 or 1 μg/ml Dox for 24 h to induce ATPase-deficient Hsc70 expression. Samples underwent immunoprecipitation with anti-Rab7 (A) or anti-Rab11 (B), and β-ENaC was detected in precipitated proteins by immunoblot using anti-V5. Input samples represent 10% of total protein subject to immunoprecipitation. Fold change in V5-β-ENaC coprecipitated with Rab7 or Rab11 was determined by densitometric analysis of precipitated V5 from 3 independent experiments. Values (means ± SE) are presented relative to control (0 μg/ml Dox). *P = 0.029 (A); in B, P = 0.27 (by t-test).

Fig. 10.

Hsc70 diminishes interaction between newly synthesized β- and α-subunits of ENaC. A: schematic diagram of the method used in B. MDCK cells with inducible expression of Hsc70-Myc/His were incubated without or with 5 μg/ml Dox for 24 h. Newly synthesized proteins underwent pulse radiolabeling and were then chased for 0–60 min; 250 μg of cell lysates were subjected to 2 consecutive immunoprecipitation steps; 10% of the proteins precipitated with anti-V5 (for β-ENaC) were removed for resolution by SDS-PAGE and PhosphorImager analysis, and the remainder were used for the 2nd immunoprecipitation step with anti-HA (for α-ENaC) and subsequent SDS-PAGE and PhosphorImager analysis. Top: fluorograms from noncontiguous lanes of the same experimental gels. Bottom: densitometric quantification of precipitated β- and α-ENaC. Values are means ± SE of 3 independent experiments.