CFTR Regulation of ENaC

The idea that cystic fibrosis (CF) results from dysregulation of ion channels, including the epithelial Na⁺ channel (ENaC), evolved from observations made before the cloning of the CF gene in 1989. Multiple laboratories had reported that protein kinase A (PKA) stimulated a Cl∼ channel called the ORCC (outward rectifying Cl^- channel) in cells from normal, but not CF, epithelial tissues (1 -3 ). We had also reported that amiloride-sensitive Na⁺ absorption was elevated in the airway epithelia of CF patients (4 -6 ). Once cloned, sequences within the CF gene identified it as a member of the ATP-binding cassette (ABC) transporter superfamily (7 ). Surprisingly, the CF gene product was found to form a Cl^- channel with distinctly different properties from the ORCC (8 ,9 ). Because ABC proteins were known to function as both transporters and regulators of other processes (10 ), and because the regulation of ORCC and amiloride-sensitive Na⁺ absorption were affected in CF, the CF gene product was named the cystic fibrosis transmembrane conductance regulator (CFTR) (11 ). Subsequently, CFTR has been reported to affect the activity of a large number of other ion channels and solute transporters (12 ). The chief interest in CFTR’ s ability to affect the function of other ion channels and transporters is the possibility that such secondary functions contribute in an organ-specific fashion to the pathogenesis of CF. The organ-specific ion transport abnormalities observed in CF are highly variable, ranging from decreased salt and water secretion in pancreatic (13 ) and bile ducts (14 ) to decreased salt absorption by sweat ductal epithelium (15 ). In order to understand how a CFTR function other than Cl^- conductance contributes to pathophysiology at an affected site, it is helpful to identify the molecular basis of the putative secondary function and define its role in normal physiology. Despite serious efforts, this level of understanding has not yet been achieved for CFTR’s apparent functional interactions with other ion channels and transporters, including ENaC. Lung disease is the primary cause of disability and death in CF patients, yet the nature of the relationship between CFTR function(s) and the maintenance of lung health remains controversial. In normal individuals, the removal of inhaled pathogens from the airway surface by mucociliary clearance (MCC) forms a major line of lung defense (16 ,17 ). In addition, the antimicrobial properties of airway surface liquid (ASL) contribute to the maintenance of a sterile airway environment (18 ,19 ). In CF patients, innate lung defense mechanisms become overwhelmed, and chronic infection and inflammation lead to the destruction of conducting airways. The precise defect in innate defense that results from CFTR mutations is a matter of intense investigation and debate. One school of thought holds that CFTR Cl^- channel activity determines the salt composition of airway surface liquid (20 ). This theory proposes that normal airway epithelia utilize CFTR as the exclusive path for Cl^- absorption to extract salt from the ASL, leaving a hypotonic luminal solution, much like a sweat duct. As a consequence, this model projects that CF airway surface liquid is relatively hypertonic. It was further hypothesized that a higher salt concentration in CF ASL interferes with the antimicrobial action of natural defensin molecules, leading to a breech in airway defense. Although this is an attractive model for the pathogenesis of CF lung disease, recent reports on the ionic composition of ASL in normal and CF using noninvasive ion-selective electrodes and fluorescent dyes have concluded that both normal and CF ASL are nearly isotonic and not different (21 ,22 ). This conclusion is compatible with independent assessments of the high water permeability of normal and CF airway epithelia, which suggest that neither epithelium can maintain a hypotonic ASL fluid (23 -25 ). An alternative hypothesis relating CFTR mutations to the development of lung disease proposes that regulation of ENaC by CFTR is required to maintain an ASL height that is adequate for MCC to proceed. MCC is a complex process, involving coordinated functions of ciliary beating, salt and water transport, and mucus secretion. Central to the MCC process is the maintenance of a low-viscosity peri ciliary liquid layer on the airway surface that enables cilia to beat effectively and propel mucus out of the lung (26 ,27 ). The depth of this periciliary liquid layer is determined by net salt and water movements across airways epithelia, and thus is strongly influenced by ion channel activity (21 ,25 ). The ion channels in the apical membrane of airway epithelia that are rate limiting for net salt movement are CFTR and ENaC (28 ,29 ). The abnormal CFTR and ENaC activities observed in CF may, therefore, account for a reduced MCC rate and the subsequent onset of airway infection. Support for this theory includes the repeated observation that Na⁺ absorption in CF airways is two- to threefold greater than in normal airways in vivo (30 ,31 ), in freshly excised tissues (5 ), and in various cultured airway epithelial preparations (32 -34 ). Recent studies utilizing highly differentiated airway epithelial cultures that develop rotational mucus transport further demonstrated that the hyperabsorption of Na⁺ by CF airway epithelia diminished the periciliary liquid layer and caused mucostasis (25 ). Therefore, the drastic consequences of CF lung disease appear to originate from the abnormal pattern of ion transport that results from mutations in the CF gene. Moreover, available data strongly suggest that negative modulation of ENaC by CFTR in human airways is a normal function of CFTR and relevant to CF lung disease pathogenesis. It is important to establish the molecular basis of this relationship between CFTR and ENaC, and methods that may help in this effort are the subject of this chapter.