The most common cystic fibrosis‐associated mutation destabilizes the dimeric state of the nucleotide‐binding domains of CFTR

Abstract

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...

An Electrostatic Interaction at the Tetrahelix Bundle Promotes Phosphorylation-dependent Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Opening

“…Inset shows single exponential fit of deactivation time course following ATP removal with a time constant of greater than 5 min. These characteristics are similar to those previously reported for the single E1371S catalytic mutant (39,40). B and C, macroscopic records showing that the E267R substitution reduced the baseline current prior to addition of PKA (units/ml) that can be detected for the E1371S-CFTR construct.…”

“…The lack of effect on the stability of the activated state is consistent with the virtually complete rescue of the ATP-dependent activity of the E267R bundle mutant that we observed when we combined this mutation with the E1371S catalytic mutation. The latter mutation abolishes ATP hydrolysis at site 2 and greatly prolongs open channel bursts by stabilizing the ATP-bound NBD dimer (39,40). The E267R/E1371S double mutant exhibited high steady-state control currents following activation by ATP and PKA, small relative activation by potentiators and very slow deactivation upon ATP washout similar to that reported for the E1371S single mutant.…”

“…E1371S-CFTR data in Refs. 39,40). We interpret these results to indicate that the E267R mutation has negligible effects on channel activity once a tight NBD dimer has been formed by ATP binding sans hydrolysis.…”

“…8). The E1371S mutation stabilizes the NBD dimer in the presence of ATP and greatly prolongs the open channel bursts (39,40). Our interest was to determine if the E267R mutation inhibited the activity of the E1371S catalytic mutant and/or destabilized the NBD dimer in the absence of ATP hydrolysis.…”

Vx-770 potentiates CFTR function by promoting decoupling between the gating cycle and ATP hydrolysis cycle

“…Mutations that disrupt this process could compromise the channel function and cause CF. For example, defects in NBD dimer formation have been proposed to account for the low open probability (Po) seen in ΔF508-and G551D-CFTR, the first and third most common CF-associated mutations (13)(14)(15). Therefore, finding small molecules (i.e., CFTR potentiators) that can improve the function of CFTR is expected to pave the way for targeted treatment in CF (16).…”