Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). CFTR is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis at its nucleotide binding domains (NBDs). G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Patients carrying the G551D mutation present a severe clinical phenotype. On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Residues G551 and G1349 are located at equivalent positions in the highly conserved signature sequence of each NBD. The physiological importance of these residues lies in the fact that the signature sequence of one NBD and the Walker A and B motifs from the other NBD form the ATP-binding pocket (ABP1 and ABP2, named after the location of the Walker A motif) once the two NBDs dimerize. Our studies show distinct gating characteristics for these mutants. The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is ∼100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg2+]. The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. G1349D-CFTR maintains ATP dependence, albeit with a Po ∼10-fold lower than WT. Interestingly, compared to WT results, the ATP dose–response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Thus, our data provide a quantitative explanation at the single-channel level for different phenotypes presented by patients carrying these two mutations. In addition, these results support the idea that CFTR's two ABPs play distinct functional roles in gating.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and ⌬F508. Previously we found that the ATP analog N 6 -(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by ϳ7-fold. Here we show that 2-deoxy-ATP (dATP), but not 3-deoxy-ATP, increases the activity of G551D-CFTR by ϳ8-fold. We custom synthesized N 6 -(2-phenylethyl)-2-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 ؎ 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of ⌬F508-CFTR by increasing its activity by 19.5 ؎ 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with ⌬F508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable. The cystic fibrosis transmembrane conductance regulator (CFTR)2 chloride channel is a major player in salt and water transport across epithelia. Like all members of the ATPbinding cassette (ABC) family, CFTR has two nucleotide-binding domains (NBDs), which contain the Walker A and B motifs and the highly conserved signature sequence. A regulatory (R) domain, unique to CFTR, needs to be phosphorylated by protein kinase A (PKA) for the channel to function. Experimental evidence suggests that the two NBDs of CFTR dimerize in a head-to-tail configuration (1), as in other ABC transporters, forming two ATP-binding pockets (ABPs) with two ATP molecules sandwiched in the interface. ABP1 is formed by the Walker A and B motifs of NBD1, and the signature sequence of NBD2 and ABP2 is formed by the Walker A and B motifs of NBD2 and the signature sequence of NBD1. Interestingly, two ABPs of CFTR assume distinct functional roles in controlling CFTR gating. Zhou et al. (2) showed that ABP2 is the site critical for channel opening by ATP, whereas the role of ABP1 is limited to help with the stabilization of the open channel conformation. Furthermore, although ABP1 seems unable to hydrolyze ATP, ATP hydrolysis at NBD2 is associat...

The results from these studies can be interpreted as an equilibrium shift toward the open-channel conformation of F508del-CFTR channels.

Non-technical summary Cystic fibrosis is a genetic disease caused by the malfunction of a chloride channel called cystic fibrosis transmembrane conductance regulator (CFTR). The most common disease-associated mutation is the deletion of the phenylalanine residue at position 508 ( F508), which result in channels with poor membrane expression and defective function. Opening of CFTR channels is controlled by ATP binding at two intracellular domains, called nucleotide-binding domains (NBDs), and subsequent NBD dimerization. Our previous studies revealed that F508-CFTR channels open very infrequently, raising the possibility that the mutation perturbs NBD dimerization although the mutation is not located near the dimer interface. In this paper, we employed a functional assay to assess the stability of the NBD dimer. Our data suggest that the F508 mutation significantly destabilizes the NBD dimer, supporting the hypothesis that the mutation disrupts the dimer interface. Our results provide structural insights that are potentially useful for drug design.Abstract The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP binding cassette (ABC) superfamily. The deletion of the phenylalanine 508 ( F508-CFTR) is the most common mutation among cystic fibrosis (CF) patients. The mutant channels present a severe trafficking defect, and the few channels that reach the plasma membrane are functionally impaired. Interestingly, an ATP analogue, N 6 -(2-phenylethyl)-2 -deoxy-ATP (P-dATP), can increase the open probability (P o ) to ∼0.7, implying that the gating defect of F508 may involve the ligand binding domains, such as interfering with the formation or separation of the dimeric states of the nucleotide-binding domains (NBDs). To test this hypothesis, we employed two approaches developed for gauging the stability of the NBD dimeric states using the patch-clamp technique. We measured the locked-open time induced by pyrophosphate (PP i ), which reflects the stability of the full NBD dimer state, and the ligand exchange time for ATP/N 6 -(2-phenylethyl)-ATP (P-ATP), which measures the stability of the partial NBD dimer state wherein the head of NBD1 and the tail of NBD2 remain associated. We found that both the PP i -induced locked-open time and the ATP/P-ATP ligand exchange time of F508-CFTR channels are dramatically shortened, suggesting that the F508 mutation destabilizes the full and partial NBD dimer states. We also tested if mutations that have been shown to improve trafficking of F508-CFTR, namely the solubilizing mutation F494N/Q637R and RI (deletion of the regulatory insertion), exert any effects on these newly identified functional defects associated with F508-CFTR. Our results indicate that although these mutations increase the membrane expression and function of F508-CFTR, they have limited impact on the stability of both full and partial NBD dimeric states for F508 channels. The structure-function insights gained from this mechanism may provide clues for future drug d...