G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects

Abstract

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 approximately 100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg(2+)]. 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 approximately 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.

Figure 1.

Cartoon depicting the location of G551 and G1349 in CFTR's two ATP-binding pockets. The G551 residue is located in the signature sequence of NBD1 while the G1349 is located in the signature sequence of NBD2. CFTR has two ATP-binding pockets (ABPs) composed by the Walker A and B motifs of one NBD and the signature sequence of the partner NBD. The ATP-binding pockets are named after the position of the Walker A motif (i.e., ABP1 is composed of the Walker A and B motifs of NBD1 and the signature sequence of NBD2). Thus the G551D mutation is located in ABP2 and the G1349D mutation is located in ABP1.

Figure 2.

G551D-CFTR and G1349D-CFTR expression in CHO cells. (A) Western blot analysis for WT-, G551D-, and G1349D-CFTR. Mature fully glycosylated CFTR and core-glycosylated CFTR are labeled as Band C and Band B, respectively. The same blot was probed with an antibody against the cytoskeletal protein vimentin as a control for protein loading. Similar results were obtained in three separate preparations. (B) Mean current densities obtained from whole-cell experiments for G551D (n = 30), G1349D (n = 29), and WT (n = 20). Columns and error bars indicate means ± SEM, * indicates P < 0.01 between G551D and G1349D.

Figure 3.

Gating of G551D-CFTR and G1349D-CFTR in excised inside-out membrane patches. (A) A recording of G551D-CFTR channel currents in an excised inside-out membrane patch. Note that after washout of ATP and PKA, the channels remained active for over 20 min. Expanded current traces show the G551D-CFTR channel activity in the presence of ATP + PKA and during washout. (B) Repeated addition and removal of ATP in the same patch did not result in significant changes of the channel activity of G551D-CFTR. (C) A recording of G1349D-CFTR channel currents in an excised inside-out membrane patch. The channels were activated with 1 mM ATP + 25 U/ml PKA. Upon removal of ATP and PKA, the current decays very rapidly. A small amount of current remains minutes after washout of ATP. (D) A recording of WT-CFTR channel currents in an excised inside-out membrane patch. (E) Comparisons of mean macroscopic current amplitude for G551D- (n = 16), G1349D- (n = 18), and WT-CFTR (n = 13). Data are presented as means ± SEM.

Figure 4.

Comparison of the mean open times of G551D-, G1349D-, and WT-CFTR. (A) Expanded single-channel current traces in the presence of 1 mM ATP for G551D-, G1349D-, and WT-CFTR. (B) Comparisons of the mean open times and single channel amplitudes (n = 6–14 for each data point). Data are presented as means ± SEM.

Figure 5.

Effect of [Mg²⁺] in the closing rate of WT and G551D-CFTR. Currents were activated by 1 mM ATP + PKA in a Mg²⁺-containing solution. (A) Withdrawal of ATP + PKA and Mg²⁺ ions results in channels closing more slowly for WT-CFTR. Note that some channels remain open for tens of seconds. (B) G551D-CFTR channel openings remain unaltered when ATP, PKA, and Mg²⁺ are removed from the perfusion solution (n = 6).

Figure 6.

Effect of ADP on G551D, G1349D, and WT-CFTR. Representative current traces for WT-CFTR (A), G551D-CFTR (B), and G1349D-CFTR (C). Currents from both WT- and G1349D-CFTR are reduced by ADP, but ADP fails to inhibit G551D-CFTR channel currents. (D) Summary of percent inhibition by 500 μM ADP for G551D- (n =10), G1349D- (n = 8), and WT-CFTR (n = 8). Data are presented as means ± SEM. * indicates P < 0.001 between G1349D and WT.

Figure 7.

Effect of AMP-PNP on G551D, G1349D, and WT-CFTR. Representative current traces for WT-CFTR (A), G551D-CFTR (B), and G1349D-CFTR (C). WT-CFTR channels were locked open by 2 mM AMP-PNP + 1 mM ATP. Neither G551D- nor G1349D-CFTR responds to AMP-PNP (n = 4 each).

Figure 8.

ATP dose–response relationship for G551D-CFTR. (A) Representative trace for G551D-CFTR in the presence of 10 mM ATP. Currents were activated with 1 mM ATP + 25 U/ml PKA (not depicted). (B) Normalized ATP dose–response relationship for G551D-CFTR. Currents at different [ATP] were normalized to the current level in the absence of ATP (washout) (n = 4–6 for each data point).

Figure 9.

ATP dose–response for G1349D-CFTR. Representative traces of WT- (A) and G1349D-CFTR (B) channels in response to 50 μM and 2.75 mM ATP. Note that the amount of current elicited by 50 μM ATP, relative to the amount of current elicited by 2.75 mM ATP, is larger for G1349D (ratio = 0.65) than for WT (ratio = 0.42) in these particular patches. (C) Normalized ATP dose–response relationship for G1349D-CFTR (red circles) and WT-CFTR (blue squares, from Zhou et al., 2006). (D) Relationships between [ATP] and single-channel Po (blue squares for WT, from Zhou et al., 2006, and red open circles for G1349D). The G1349D Po was calculated under the assumption that the maximal Po is 10 times lower than WT Po.

Figure 10.

(A) Scheme A: a CFTR gating model expanded from one proposed by Vergani et al. (2003). C, ATP·C, and ATP·C·ATP are closed states with no ATP bound, one ATP molecule bound to ABP1, and two ATP molecules bound, respectively. O_2ATP is the open state with two bound ATP molecules. Kd₁ and Kd₂ are the dissociation constants of ATP for ABP1 and ABP2, respectively. k _O(2ATP) is the opening rate and k _C(ATP) is the closing rate of the channel via hydrolysis. The gray arrow, representing the nonhydrolytic closing step, has been ignored in our calculation because it is exceedingly small compared with the closing rate of the hydrolytic closing (same applies to Figure 11). According to this model, the channel can open only after the two binding sites are occupied by ATP. (B) ATP dose response for WT-CFTR channels (red squares) and the simulated curve (blue line) obtained with Scheme A and the parameters summarized in Table I. (C) Normalized ATP dose response for G1349D-CFTR channels (red circles) and different simulated results obtained with Scheme A. The green line represents a fit with the same parameters as WT except for a reduced opening rate. Modification of Kd₁ up to 1,000-fold (brown line) did not reproduce the leftward shift. The red line represents simulated data when the ATP affinity at ABP2 is increased by fivefold (parameters in Table I).

Figure 11.

(A) Scheme B: an expanded CFTR gating model proposed in Zhou and Hwang (2007). C, ATP·C, C·ATP, and ATP·C·ATP are closed states with no ATP bound, one ATP bound to ABP1, one ATP bound to ABP2, and both ABP1 and ABP2 occupied, respectively. O_1ATP and O_2ATP represent open states with only ABP2 occupied by ATP or both ABP1 and ABP2 occupied by ATP, respectively. O_SPT0 and O_SPT1 are open states in the ATP-independent component with no ATP or one ATP bound to ABP1, respectively. Kd₁ and Kd₂ are the dissociation constants of ATP for ABP1 and ABP2, respectively. k _{O (1ATP)} is the opening rate with ATP bound at ABP2 (from C·ATP to O_ATP1); k _{O (2ATP)} is the opening rate with ATP binding to ABP1 and ABP2 (from ATP·C·ATP to O_ATP2). k _C(ATP) is the transition rate from open to closed state in the hydrolytic pathway both without and with ATP binding to ABP1 (from O_ATP1 to C and from O_ATP2 to C). k _O(SPT) and k _C(SPT) are the opening and closing rates for spontaneous ATP-independent gating. (B) Dose–response relationships for WT (red squares) and G1349-CFTR (red circles) and calculated data based on Scheme B and the parameters summarized in Table II