Multiple Membrane-Cytoplasmic Domain Contacts in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mediate Regulation of Channel Gating

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) 3 , the mutation of which causes cystic fibrosis (CF), belongs to the superfamily of ATP-binding cassette (ABC) proteins but functions as an ion channel rather than an active transporter. The chloride channel activity is crucial for maintaining salt and fluid homeostasis in epithelial tissues (1). In patients with CFTR mutations that compromise its maturation or channel activity, the airway surface liquid volume is diminished, impeding mucociliary clearance (2, 3). The absence of functional CFTR also impairs submucosal gland secretion (4).Like many other ABC family proteins, CFTR (also known as ABCC7) contains two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs), with an additional unique R domain (Fig. 1). While many ABC proteins are multisubunit proteins composed of two identical NBDs and MSDs, CFTR is a single polypeptide containing two distinct NBDs and MSDs. The proper folding of the individual domains and the interactions between these domains during or after protein synthesis are essential for CFTR assembly, a process that is inefficient with the majority of CFTR being degraded at the endoplasmic reticulum by the proteasome (5, 6). The most prevalent CF-causing mutation is the deletion of a phenylalanine at position 508 (⌬F508). Recent studies suggest that the folding kinetics of NBD1 and the interdomain interactions between MSDs and the NBDs are disrupted by this mutation (7-9), although the crystal structures of isolated wild-type and mutant NBD1 show no major alteration in its overall threedimensional structure (10).Control of CFTR channel activity is modulated by the phosphorylation of the R domain by protein kinase A, which allows the regulation of gating by ATP binding at the NBD1/NBD2 interface. Stable binding of ATP at NBD1 and binding and hydrolysis of ATP at NBD2, together with R domain phosphorylation, may alter allosteric interactions between these domains and impact the channel gating cycle (11-13). Although considerable progress has been made toward understanding the integrated control of CFTR channel gating by phosphorylation and ATP binding/hydrolysis (12), details at the level of interactions of specific secondary and tertiary struc-

Mutation-Specific Potency and Efficacy of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Potentiators

“…ICLs now are considered particularly interesting because they may couple changes in NBD conformation to movements of transmembrane segments, thus allowing gating of the CFTR pore (Mendoza and Thomas, 2007;He et al, 2008;Mornon et al, 2008;Serohijos et al, 2008). In the present study, we have investigated the ability of CFTR potentiators to overcome the gating defect caused by ICL mutations.…”

C Terminus of Nucleotide Binding Domain 1 Contains Critical Features for Cystic Fibrosis Transmembrane Conductance Regulator Trafficking and Activation

“…Contacts between the different domains of CFTR are essential to the maturation and activity of CFTR. For instance, the contacts established between the intracellular loops of the membrane spanning domains and the NBDs (28,31), in which Phe 508 participates, are critical for the interdomain assembly and for the regulation of channel gating (31)(32)(33). The dimeric organization of the two NBDs is also a key feature for an optimal catalytic activity of CFTR (34).…”

Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

“…The pore formed by TMD1 and TMD2 is gated by binding/hydrolysis of ATP at the interface of NBD1 and NBD2 and regulated by R domain phosphorylation/dephosphorylation (11,12). Although CLs have been found to play a central role in channel gating (6,7,9,10,13,14), it is largely unknown how CLs couple ATP actions at NBDs to gating rearrangements of TMDs and how R domain phosphorylation by cAMP-dependent PKA mediates the coupling.…”

“…Both TMDs consist of six membrane-spanning helixes that are extended to the intracellular side to form four long cytoplasmic loops (CLs) (3). These CLs interact not only with NBD1 and NBD2 but also with the R domain (3)(4)(5)(6)(7)(8)(9)(10). The pore formed by TMD1 and TMD2 is gated by binding/hydrolysis of ATP at the interface of NBD1 and NBD2 and regulated by R domain phosphorylation/dephosphorylation (11,12).…”