The cystic fibrosis transmembrane conductance regulator impedes proteolytic stimulation of the epithelial Na+ channel

Abstract

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) that prevent its proper folding and trafficking to the apical membrane of epithelial cells. Absence of cAMP-mediated Cl(-) secretion in CF airways causes poorly hydrated airway surfaces in CF patients, and this condition is exacerbated by excessive Na(+) absorption. The mechanistic link between missing CFTR and increased Na(+) absorption in airway epithelia has remained elusive, although substantial evidence implicates hyperactivity of the epithelial Na(+) channel (ENaC). ENaC is known to be activated by selective endoproteolysis of the extracellular domains of its α- and γ-subunits, and it was recently reported that ENaC and CFTR physically associate in mammalian cells. We confirmed this interaction in oocytes by co-immunoprecipitation and found that ENaC associated with wild-type CFTR was protected from proteolytic cleavage and stimulation of open probability. In contrast, ΔF508 CFTR, the most common mutant protein in CF patients, failed to protect ENaC from proteolytic cleavage and stimulation. In normal airway epithelial cells, ENaC was contained in the anti-CFTR immunoprecipitate. In CF airway epithelial cultures, the proportion of full-length to total α-ENaC protein signal was consistently reduced compared with normal cultures. Our results identify limiting proteolytic cleavage of ENaC as a mechanism by which CFTR down-regulates Na(+) absorption.

FIGURE 1.

CFTR interacts with ENaC and decreases ENaC-mediated I_Na. A, CFTR and ENaC associate with each other when expressed in Xenopus oocytes. Oocytes were injected with rat α-, β-, and HA/V5-tagged γ-ENaC cRNAs in the absence or presence of CFTR cRNA. CFTR was immunoprecipitated (IP) with rabbit anti-CFTR antibody 155, and HA/V5-tagged γ-ENaC was immunoprecipitated with rabbit anti-HA antibody (Abcam ab9110). Immunoprecipitated proteins were detected by Western blotting using anti-CFTR mAb 596 and anti-HA mAb (Covance HA.11). B, CFTR coexpression decreases ENaC-mediated I_Na. Current was recorded from oocytes injected with ENaC cRNAs (0.3 ng/each) alone or with CFTR cRNA (1–2 ng). Amiloride was removed for 30 s to measure basal I_Na. Trypsin (2 μg/ml) was applied for 5 min, and I_Na was determined again (n = 84 oocytes in each group, from 14 separate batches of oocytes). ^a, trypsin-stimulated versus basal I_Na (p < 0.001); ^b, ENaC + CFTR basal versus ENaC basal I_Na (p < 0.01); ^c, CFTR + ENaC trypsin-stimulated versus ENaC trypsin-stimulated I_Na. Analysis of variance with Tukey's test was performed. C, CFTR coexpression increases stimulation of ENaC by trypsin. ENaC I_Na reported in B was analyzed as -fold trypsin stimulation. In oocytes expressing ENaC, I_Na was increased by 5.16 ± 0.35-fold. In oocytes coexpressing ENaC and CFTR, trypsin increased I_Na by 8.69 ± 0.46-fold (n = 84 oocytes in each group, from 14 separate batches of oocytes). ^a, p < 0.001 by unpaired t test. D, CFTR coexpression decreases ENaC whole cell open probability (P_o). Oocytes were injected with cRNA for wild-type α- and γ-ENaCs and S518C β-ENaC (0.3 ng each) alone or the same ENaC subunit combination with CFTR (1 ng). Amiloride was removed for 30 s to measure basal I_Na. MTSET (1 mm) was applied for 5 min, and I_Na was determined again. Whole cell P_o was calculated from the ratio of basal I_Na to MTSET-stimulated I_Na (n = 44 oocytes in each group, from 14 separate batches of oocytes). ^a, p < 0.001 by unpaired t test.

FIGURE 2.

Specific inhibition of ENaC proteolysis and I_Na by CFTR. A, ENaC was expressed in Xenopus oocytes (as described for Fig. 1B) alone, with matriptase (Mtrp), and with both matriptase and CFTR. Basal I_Na was increased by matriptase and not further stimulated by exogenous trypsin (2 μg/ml). CFTR prevented stimulation of basal I_Na and restored sensitivity to exogenous trypsin. B, CFTR prevents proteolytic cleavage of α- and γ-ENaCs by matriptase. Oocytes were injected with all three ENaC subunits, whereby either the α- or γ-subunit was tagged at the N and C termini with HA and V5 epitopes, respectively. Similar experiments were performed with coexpression of matriptase and CFTR or MRP1. Oocyte lysates were analyzed by Western blotting using anti-V5 mAb to detect ENaC, mAb 596 to detect CFTR, and mAb 42.4 to visualize expression of MRP1. As a control for loading of equal amounts of proteins, actin was detected with anti-actin mAb. C, experiments were similar to those described for A except that CFTR was replaced by MRP1. Matriptase coexpression was associated with large basal I_Na that was unaffected by trypsin, and coexpression of MRP1 did not alter this result. ^a, trypsin-stimulated I_Na versus basal I_Na (p < 0.001); ^b, basal I_Na of ENaC + Mtrp or ENaC + Mtrp + MRP1 versus basal I_Na of ENaC alone (p < 0.01). Analysis of variance with Tukey's test was performed (A and C, n = 21–22 oocytes combined from five batches).

FIGURE 3.

ΔF508 CFTR does not diminish proteolysis of ENaC. A, Xenopus oocytes were injected with all three ENaC subunits, whereby either the α- or γ-subunit was tagged at the N and C termini with HA and V5 epitopes, respectively. CFTR or ΔF508 CFTR was coexpressed with ENaC and matriptase (Mtrp). Oocyte lysates were analyzed by Western blotting as described for Fig. 3. B, wild-type CFTR but not ΔF508 CFTR interferes with stimulation of ENaC by matriptase. I_Na of (ENaC + Mtrp)-expressing oocytes was not further stimulated by a 5-min exposure to trypsin (2 μg/ml). Coexpression of wild-type CFTR but not ΔF508 CFTR significantly decreased basal I_Na, and the reduced current was partially recovered by trypsin. Data were combined from three batches of oocytes (n = 16–17). ^a, basal I_Na of ENaC + Mtrp + CFTR versus basal I_Na of ENaC + Mtrp or ENaC + Mtrp + ΔF508 CFTR (p < 0.01); ^b, trypsin-stimulated I_Na versus basal I_Na (p < 0.05). Analysis of variance with Tukey's test was applied.

FIGURE 4.

Endogenous CFTR and ENaC co-immunoprecipitate, and CFTR impedes proteolysis of ENaC in primary HAE cultures. A, α-ENaC co-immunoprecipitates with CFTR in primary airway cells (HAE). CFTR was immunoprecipitated (IP) with rabbit anti-CFTR Ab and pulled down with protein A-agarose beads, and associated α-ENaC was detected with mAb UNC1 19.2.1. CFTR was visualized with mAb 596. Control lysates were treated identically but did not contain anti-CFTR Ab. 2% of immunoprecipitation inputs were loaded. B, ENaC is less cleaved in normal (NL) primary HAE cultures than in CF cultures (ΔF508/ΔF508). Primary human HAE cells were grown on an air-liquid interface until well polarized, and lysates were analyzed by Western blotting with anti-α-ENaC mAb UNC1 19.2.1. Three different cultures with cells derived from three different normal or CF individuals were analyzed. CFTR was visualized with mAb 596 after immunoprecipitation with rabbit polyclonal Ab.