A Conserved Region of the R Domain of Cystic Fibrosis Transmembrane Conductance Regulator Is Important in Processing and Function

Abstract

The R domain of cystic fibrosis transmembrane conductance regulator (CFTR) connects the two halves of the protein, each of which possess a transmembranespanning domain and a nucleotide binding domain. Phosphorylation of serine residues, which reside mostly within the C-terminal two-thirds of the R domain, is required for nucleotide-dependent activation of CFTR chloride channel activity. The N terminus of the R domain is also likely to be important in CFTR function, since this region is highly conserved among CFTRs of different species and exhibits sequence similarity with the "linker region" of the related protein, P-glycoprotein. To date, however, the role of this region in CFTR channel function remains unknown. In this paper, we report the effects of five disease-causing mutations within the N terminus of the CFTR-R domain. All five mutants exhibit defective protein processing in mammalian HEK-293 cells, suggesting that they are mislocalized and fail to reach the cell surface. However, in the Xenopus oocyte, three mutants reached the plasma membrane. One of these mutants, L619S, exhibits no detectable function, whereas the other two, D614G and I618T, exhibit partial activity as chloride channels. Single channel analysis of these latter two mutants revealed that they possess defective rates of channel opening, consistent with the hypothesis that the N terminus of the R domain participates in ATP-dependent channel gating. These findings support recent structural models that include this region within extended boundaries of the first nucleotide binding domain.

Stimulatory and inhibitory protein kinase C consensus sequences regulate the cystic fibrosis transmembrane conductance regulator

“…The inhibitory site T682 is present only in mammalian CFTRs, therefore the absence of this site may contribute to the stronger activation of amphibian CFTR by PKC. T582 and T604 are located in NBD1 near the R domain, and S641 is in a highly conserved region called RD1, which is sensitive to mutagenesis (29). Recent NBD structures from bacterial ABC transporters suggest they may function as dimers, with the LSGGQ motif (ABC transporters signature) from one NBD facing the ATP-binding site of the other NBD (reviewed by ref.…”

Disease-associated Mutations in the Extracytoplasmic Loops of Cystic Fibrosis Transmembrane Conductance Regulator Do Not Impede Biosynthetic Processing but Impair Chloride Channel Stability

“…Other single residue substitutions alter specific properties of the CFTR ion channel (14). For example a nucleotide binding domain mutation, G551D, precludes virtually all activity and R-domain mutations have a variety of effects (15). Substitutions in membrane-spanning sequences may alter conductance, selectivity, occupancy, or gating of the pore (16).…”

Lung Infections Associated with Cystic Fibrosis

“…According to the same model, when the R domain becomes phosphorylated, it either dissociates from the NBD or interacts with the NBD in a different way, such that the ATP binding site of NBD-1 is available. Further evidence for the important role of the R domain in channel activity was obtained through the analysis of R-domain point mutants, which demonstrated either reduced channel activity or complete loss of activity (219). Deletions in the R domain result in regulatory defects (180).…”