Function of the R Domain in the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel

Abstract

For a cystic fibrosis transmembrane conductance regulator (CFTR) channel to enter its open state, serine residues in the R domain must be phosphorylated by cAMP-dependent protein kinase, and intracellular ATP must bind to the nucleotide-binding folds and subsequently be hydrolyzed. CFTR with its R domain partially removed, ⌬R(708 -835)-CFTR, forms a chloride channel that opens independently of protein kinase A phosphorylation, with open probability approximately one-third that of the wild type CFTR channel. Deletion of this portion of the R domain from CFTR alters the response of the channel to 5-adenylylimidodiphosphate, pyrophosphate, and vanadate, compounds that prolong burst duration of the wild type CFTR channel but fail to do so in the ⌬R-CFTR. In addition, the addition of exogenous unphosphorylated R domain protein, which blocks the wild type CFTR channel, has no effect on the ⌬R-CFTR channel. However, when the exogenous R domain is phosphorylated, significant stimulation of the ⌬R-CFTR channel results; Rich et al. (9) demonstrated that deletion of 128 amino acids from the R domain of CFTR, ⌬R(708 -835), leads to a chloride channel that opens without PKA phosphorylation. This portion of the R domain corresponds to sequences that are not conserved in related proteins, i.e. MDR1 and STE6 (9 -11). The portion of the R domain that remains in ⌬R-CFTR corresponds to amino acids that are present in other ATP-binding cassette transporters between the predicted first nucleotide binding fold and the predicted second membrane-spanning domain (9, 11). Our studies show that unphosphorylated R domain protein synthesized in vitro (amino acids 590 -858) interacts specifically with CFTR to inhibit chloride conductance in a phosphorylationdependent manner (12). These studies were interpreted to be consistent with the hypothesis that the putative "gating particle" of the CFTR channel resides within the R domain.The first and second NBF of CFTR share sequence similarity in certain conserved regions such as Walker A and Walker B motifs, but the overall amino acid homology between the two NBFs of CFTR is only ϳ30%. Functional studies of CFTR containing site-directed mutations in NBF1 and NBF2 suggest that the two NBFs have different roles in the gating of the CFTR channel (13)(14)(15). Mutations predicted to interfere with nucleotide hydrolysis in the first NBF reduce the channel opening rate, but the corresponding mutations in the second NBF result in prolonged channel openings (15). The use of compounds that alter the ATP hydrolysis cycle of CFTR, such as 5Ј-adenylylimidodiphosphate (AMP-PNP), PP i , and vanadate (VO 4 ), provided evidence that hydrolysis of ATP is not only required for channel opening but also is involved in channel closure from the bursting state (16 -19). Based on these studies, it was proposed that ATP hydrolysis at NBF1 initiates a burst of activity and that hydrolysis at NBF2 terminates a burst of open events (15).This dual regulatory mechanism of the CFTR channel (PKA phosphorylation of the R domain ...

A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution

“…The Cterminal half of exon 13 appears to be the functional R ''regulatory'' domain (9,10). Consistent with this, deletion of residues 708-835 produces a CFTR channel with properties like those of wild-type CFTR, suggesting that residues 708-835 form a functional R domain that can be deleted (9,(12)(13)(14). More extensive deletions (residues 590-835, 656-835, 676-835, or 681-835) disrupted function (9), probably because they disrupt a portion of NBD1.…”

“…CFTR-⌬R͞S660A lacks the serines (660, 737, 795, and 813) shown to be responsible for the majority of PKA-stimulated channel activity (6,15,16,19). This channel differs from wild-type CFTR in two important ways: it has a low level of constitutive Cl Ϫ channel activity in the absence of PKA-dependent phosphorylation, and its activity is not altered by addition of PKA (9,(12)(13)(14).…”

CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

“…According to helical-wheel analysis, residues 733-739 and 761-777 (depending on the side chain conformation of Arg766) may also form amphipathic helices, and these might bind the same NBD1 surface as the regulatory extension, or other NBD1 surface(s), in a dynamic equilibrium. Additional residues of the R region, including 767-780, which interact with ATP-bound NBD1 independently of phosphorylation, may bind this same NBD1 surface or other NBD1 surface(s) 42 . Further NMR studies are currently in progress that focus on NBD1 to clarify the sites of R-region interactions.…”

Phosphorylation-induced Conformational Changes of Cystic Fibrosis Transmembrane Conductance Regulator Monitored by Attenuated Total Reflection-Fourier Transform IR Spectroscopy and Fluorescence Spectroscopy

“…PKA phosphorylation relieves this inhibition. On the other hand, it has been shown that exogenous phosphorylated R domain (either residues 590 -858, 645-834, or 708 -831) increases the open probability of a CFTR channel construct missing residues 708 -835 from the R domain (7,10,11).…”

“…It is believed that the R domain combines both inhibitory and stimulatory effects. Effectively, deletion of the residues 708 -835 from the R domain (6,7), and even of the residues 760 -783 (8), generates constitutively active channels. Moreover, overexpression or addition of the unphosphorylated R domain, encompassing residues 590 -858, inhibits chloride transport (9).…”