Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channel by Its R Domain

Abstract

Since the sequence of the CFTR 1 Cl Ϫ channel was discovered, the function of its R domain has been puzzling. CFTR is a member of the ATP-binding cassette (ABC) transporter family, and it contains the features characteristic of this family: two nucleotide binding domains (NBDs) and two membrane-spanning domains (MSDs) (1). Yet in addition, CFTR also contains the R domain, a unique sequence not found in other ABC transporters or any other proteins. The R domain serves as the major physiologic regulator of the CFTR Cl Ϫ channel (2, 3). Upon elevation of cAMP levels, cAMP-dependent protein kinase (PKA) phosphorylates the R domain allowing ATP to open and close the channel. Yet how phosphorylation activates the channel is not well understood. Some models propose that the R domain prevents the channel from opening and that phosphorylation relieves this inhibition. Other models suggest that phosphorylation of the R domain stimulates activity. Here we briefly review current knowledge that may help explain the function of this interesting domain. Sequence and Boundaries of the R DomainThe R domain was originally defined as those residues encoded by exon 13 (aa 590 -830) ( Fig. 1) (1). Although the precise boundaries of the R domain remain uncertain, recent studies suggest that the N-terminal portion of exon 13 is actually part of NBD1, whereas the C-terminal portion constitutes the structural and functional "R domain." Supporting this conclusion, the N-terminal portion of exon 13 has sequence similarity with NBDs of other ABC transporters (4, 5). More recently, crystal structures were solved for the NBDs of the ABC transporters HisP, MalK, and Rad50 (6 -8). In CFTR, residues analogous to those NBDs would extend to approximately aa 642. Experimental evidence for the boundaries came with the demonstration that deletion of aa 708 -835, but not deletions extending further in the N-terminal direction, produced a channel that was processed correctly, opened in the presence of ATP, and had conductive properties like those of wild-type CFTR (4, 9). In addition, severing CFTR after residues 633 or 835 and coexpressing the two halves in Xenopus oocytes produced functional channels, whereas severing the channel between exons 12 and 13 abolished function (10, 11). All these data suggest that the N-terminal R domain boundary begins between residues 634 and 708 and that the C-terminal boundary ends in the region of residue 835.The most striking feature of the R domain is the presence of multiple consensus PKA phosphorylation sites that are highly conserved across species (Fig. 1). There is little other sequence similarity in the R domain, unlike the substantial sequence conservation throughout the rest of CFTR. We are not aware of any other conserved protein motifs in the R domain, although there is a relatively high percentage (28%) of charged residues. Sixteen of the 24 basic residues are in PKA consensus sites. Several of the acidic residues are clustered within 817-838 (12). Phosphorylation of the R DomainPKA is the primary kinas...

Regulatory R region of the CFTR chloride channel is a dynamic integrator of phospho-dependent intra- and intermolecular interactions

“…The longest and strongest binding segment of R region for NBD1 comprises residues 748-778, consistent with data showing that lack of segment 760-783 also results in a constitutively active channel (36). The phosphorylation dependence of the binding supports the model, which is shown in Fig.…”

Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia

“…Thus, we do not know how the acidic cluster regulates channel gating, but we speculate that this cluster might bind the highly charged R domain via an electrostatic interaction. The effect of the acidic cluster on opening rate (interburst interval) but not burst duration is consistent with the way that other R domain interventions influence gating (42).…”

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

“…However, the deletion of the R domain does not affect CFTR expression (10, 24 -27). Moreover, the location of the R domain was found not to be essential for the function (28). Finally, the R domain is unstructured and disordered (29,30).…”