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Key Event Title
Cystic Fibrosis Transmembrane Regulator Function, Decreased
|Level of Biological Organization|
Key Event Components
|chloride channel activity||decreased|
Key Event Overview
AOPs Including This Key Event
|All life stages||High|
Key Event Description
The cystic fibrosis transmembrane regulator (CFTR) is a multi-domain membrane protein that belongs to the large family of adenine nucleotide binding cassette transporters consisting of two transmembrane domains, two nucleotide binding domains (NBDs) and a unique regulatory domain (Riordan, 2008). It is an integral membrane glycoprotein that functions as cAMP-activated and phosphorylation-regulated Cl– channel at the apical membrane of epithelial cells (Farinha et al., 2013). In respiratory epithelia, CFTR is the major Cl– channel that mediates fluid and electrolyte transport, and CFTR function is critical to normal ASL homeostasis. Exposure to inhaled oxidants, such as ozone and cigarette smoke, leads to decreased CFTR gene and protein expression as well as CFTR internalization, thereby reducing or abolishing short-circuit currents (Qu et al., 2009; Cantin et al., 2006a; Cantin et al., 2006b; Clunes et al., 2012; Sloane et al., 2012; Rasmussen et al., 2014). Reduced CFTR gene transcription rates were mechanistically linked to mobilization of intracellular Ca2+, resulting in decreased mRNA and protein expression, presumably in a protein kinase-dependent manner (Bargon et al., 1992a; Bargon et al., 1992b). Cigarette smoke exposure of primary human bronchial epithelial cells at the air-liquid interface was shown to rapidly increase intracellular Ca2+, followed by a decrease in cell surface CFTR expression (Rasmussen et al., 2014). Of note, this decrease by CFTR internalization was subsequently linked to decreased active Cl– transport and a reduction in ASL height/volume (Clunes et al., 2012). Similarly, treatment with pyocyanin, a redox-active virulence factor secreted by Pseudomonas aeruginosa which commonly infects the airways of cystic fibrosis patients, increased hydrogen peroxide levels in CFBE41o- bronchial epithelial cells in a dose- and time-dependent manner, leading to oxidation of the cytosol and inhibited forskolin-stimulated ion transport (Schwarzer et al., 2008). Other possible mechanisms of acquired CFTR dysfunction include direct covalent modification of the protein by cigarette smoke and acrolein (Raju et al., 2013; Raju et al., 2016a) or modulation of channel open probability (Zhang et al., 2013; Woodworth, 2015).
How It Is Measured or Detected
In cystic fibrosis patients, who carry a defect in the CFTR gene, the determination of the residual levels of normal, full-length CFTR transcripts may have some clinical utility in estimating CFTR function (Amaral et al., 2004). Moreover, decreased CFTR mRNA and protein expression were previously shown to result in reduced CFTR-mediated Cl− transport (Cantin et al., 2006a; Cantin et al., 2006b; Clunes et al., 2012; Sloane et al., 2012; Rasmussen et al., 2014). Therefore, measuring decreased CFTR function could be achieved by a combination of multiple techniques. For example, decreased expression of CFTR mRNA and protein in cells and tissues can be directly assessed using RT-PCR, Northern blot and Western blot or immunocyto-/histochemical methods, respectively. Of note, CFTR gene expression is generally low as is protein abundance, and protein detection methods in general perform more robustly in cultured cells than in native tissues (Farinha et al., 2004). Other, less frequently used methods include cell surface biotinylation, enabling a distinction between intracellular and cell surface forms of the protein if one wishes to study plasma membrane-expressed CFTR. In vitro or ex vivo, CFTR channel function can be assessed in real-time using patch-clamping of whole (single) cells or cell patches. In the whole-cell patch-clamp approach, current flow through CFTR can be assessed by voltage-clamp, whereas current-clamping provides insights into the effects of CFTR currents on membrane voltage (Sheppard et al., 2004). Measuring the efflux of radiolabeled tracers is another means of studying CFTR channel function, permitting a higher throughput than patch-clamping (Norez et al., 2004). The most commonly used method to study CFTR ion transport, however, utilizes the Ussing chamber to measure transepithelial voltage or “active transport potential” and short-circuit current (Li et al., 2004). In vivo, CFTR dysfunction is demonstrated by the chloride sweat test, the gold standard diagnostic tool for cystic fibrosis. The sweat test should be performed according to clinical guidelines using the Gibson and Cooke technique (also known as quantitative pilocarpine iontophoresis sweat test) (Farrell et al., 2017; Smyth et al., 2014). As a complementary diagnostic measure, nasal potential difference (NPD) can be assessed to gauge net transepithelial active ion transport and epithelial ion conductance (Schüler et al., 2004). An entire issue of the Journal of Cystic Fibrosis dedicated to the Virtual Repository of the CFTR Working Group, including the description of consensus research methods, selected principles, techniques and reagents for the assessment of CFTR expression and function is available here: https://www.sciencedirect.com/journal/journal-of-cystic-fibrosis/vol/3/suppl/S2
Domain of Applicability
Phylogenetic analysis of CFTR DNA sequences across multiple species suggests a close evolutionary relationship between human and primate CFTR, followed by rabbit, guinea pig, equine, ovine, and bovine CFTR, whereas rodent CFTR DNA largely diverges from the human DNA (Chen et al., 2001). Of note, CFTR ion permeability differs from species to species (Higgins, 1992). For example, murine CFTR displays reduced channel activity compared with its human counterpart, while ovine CFTR exhibits higher ATP sensitivity, greater single-channel conductance and larger open probability than human CFTR. Moreover, sensitivity to pharmacological agents able to potentiate or block CFTR gating varies greatly from species to species (Bose et al., 2015). Therefore, results from animal studies are not easily and directly transferable to human.
CFTR dysfunction as a consequence of inherited CFTR gene defects is studied in pediatric as well as adult cystic fibrosis patients. Acquired CFTR dysfunction following inhalation exposures (e.g. to cigarette smoke) may also apply to both pediatric and adult populations, depending on the setting and type of exposure. To our knowledge, the role of gender has not been systematically evaluated in acquired CFTR dysfunction. It is thought that the observed suppression of CFTR expression and impairment of CFTR function in cigarette smokers is a contributing factor to the pathogenesis of chronic obstructive pulmonary disease (COPD) (Dransfield et al., 2013; Raju et al., 2016b). The main risk factor for COPD is cigarette smoking, and COPD is more common in men than in women, which may be directly related to the higher prevalence of smoking in men, although this gender gap is closing (Hitchman and Fong, 2011; Ntritsos et al., 2018; Syamlal et al., 2014). Nevertheless, the available clinical evidence in support of this AOP suggests that there is no remarkable gender difference.
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