Role of Protein Kinase A-Mediated Phosphorylation in CFTR Channel Activity Regulation

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel expressed on the apical membrane of epithelial cells, where it plays a pivotal role in chloride transport and overall tissue homeostasis. CFTR constitutes a unique member of the ATP-binding cassette transporter superfamily, due to its distinctive cytosolic regulatory (R) domain carrying multiple phosphorylation sites that allow the tight regulation of channel activity and gating. Mutations in the CFTR gene cause cystic fibrosis, the most common lethal autosomal genetic disease in the Caucasian population. In recent years, major efforts have led to the development of CFTR modulators, small molecules targeting the underlying genetic defect of CF and ultimately rescuing the function of the mutant channel. Recent evidence has highlighted that this class of drugs could also impact on the phosphorylation of the R domain of the channel by protein kinase A (PKA), a key regulatory mechanism that is altered in various CFTR mutants. Therefore, the aim of this review is to summarize the current knowledge on the regulation of the CFTR by PKA-mediated phosphorylation and to provide insights into the different factors that modulate this essential CFTR modification. Finally, the discussion will focus on the impact of CF mutations on PKA-mediated CFTR regulation, as well as on how small molecule CFTR regulators and PKA interact to rescue dysfunctional channels.

Keywords: F508del-CFTR mutation; VX770; VX809; cystic fibrosis; cystic fibrosis transmembrane conductance regulator; phosphorylation; protein kinase A.

Figure 1

The impact of PKA-mediated phosphorylation on CFTR channel. The cystic fibrosis transmembrane conductance regulator (CFTR) structure consists of two membrane spanning domains, two nucleotide-binding domains (NBD1 and NBD2), and the unique cytoplasmic regulatory (R) domain. Among these, the latter represents a critical site of channel regulation due to its enrichment in protein kinase A (PKA) consensus motifs, with multiple serines and threonines as phosphorylation targets. Moreover, there is an additional phosphorylation site in the regulatory insertion (RI) segment of NBD1. In the closed CFTR state (left channel), the NBD1 interacts with the R domain creating steric hindrances which prevent it from dimerization with NBD2. Upon PKA-dependent phosphorylation (right channel), large conformational changes occur that decrease this interaction leading to the release of the R region from its inhibitory position and allow NBDs dimerization, ultimately ATP binding and CFTR-mediated chloride secretion. Thus, the open probability of the channel is dependent on the access of PKA to the main phospho-sites (S422, S660, S737, S768, S795, and S813). Phospho-sites critical for CFTR channel activation are shown in red. Phosphoserines S737 and S768 are shown in yellow since they have been shown to either activate or inhibit CFTR gating. The role of the major PKA consensus sites in the regulation of CFTR function is detailed in Table 1.