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.
Highlights
Elevated epithelial Naϩ absorption was first detected by in vivo assays of nasal and bronchial epithelial potential difference in cystic fibrosis (CF)2 patients [1], and it has been shown to contribute to depletion of airway surface liquid (ASL) in well
The pool of ␥-epithelial Naϩ channel (ENaC) found in the proteins immunoprecipitated by anti-CFTR antibody showed fewer signs of cleavage by endogenous oocyte proteases, and functional assays clearly indicated that ENaC coexpressed with CFTR was protected from proteolytic activation
We were able to bridge our observations in the oocyte expression system to the regulation of ENaC in human tissue by demonstrating that CFTR and ENaC co-immunoprecipitate in normal human primary airway epithelia (Fig. 4)
Summary
Elevated epithelial Naϩ absorption was first detected by in vivo assays of nasal and bronchial epithelial potential difference in cystic fibrosis (CF)2 patients [1], and it has been shown to contribute to depletion of airway surface liquid (ASL) in well. 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. This reduction in basal INa and increased -fold trypsin stimulation indicate reduced proteolytic activation of ENaC on the surface of oocytes expressing CFTR.
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