Optimization of the degenerated interfacial ATP binding site improves the function of disease-related mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.

FIGURE 1.

Effects of Trp-401 mutations on the lock-open time of CFTR. A, a macroscopic current recording of G551D channels in response to ATP or PATP as marked. Top left, the structural formula of PATP and a graphic showing that the NBD1 core subdomain is the site of action of PATP. D, W, and Y, indicate single-letter amino acid codes. P, PATP. B, current traces showing WT- or W401G-CFTR channels locked open by PP_i with ATP or PATP. The current decay following the removal of ligands was fitted with a single exponential functions (red, green, and light blue curves). C, comparison of current decay traces shown in B. Red, WT/ATP + PP_i; green, W401G/ATP + PP_i; light blue, WT/PATP + PP_i. D, the current relaxation time constant upon washout of PP_i + ATP (blue bars) or PP_i + PATP (green bars) for CFTR channels with different Trp-401 mutations. The number above each bar represents the number of patches.

FIGURE 2.

W401Y and W401F mutations confer ATP-dependent activation of G551D channels. A, application of ATP or PATP significantly increased currents of W401Y,W401F/G551D channels. Insets, current relaxation traces recorded after the removal of ATP (blue box) or PATP (green box) for W401F/G551D-CFTR. B, the ratio of ATP- or PATP-induced current over the basal current for G551D channels with or without Trp-401 mutations. C, effects of W401Y,W401F mutations on the relaxation time constants upon removal of ATP or PATP as shown in panel A. Note that for G551D-CFTR (Trp-401), no current decay was seen because the effect of ATP was negligible. The number above each bar represents the number of patches.

FIGURE 3.

The role of NBD2 signature motif in mediating ATP response of optimized G551D channels. A, the S1347G mutation diminished the response of W401F/G551D channels to ATP or PATP. B, incorporating the H1348G mutation into G551D-CFTR conferred responsiveness to ATP. C, H1348G enhanced the response of W401F/G551D-CFTR to ATP. D, histogram summarizing the ratio of ATP (blue bars)- or PATP (green bars)-induced current over basal current for W401F/G551D channels combined with a mutation in the NBD2 signature motif as marked. Dashed lines, the ratio of I_ATP/I_Basal (blue) and I_PATP/I_Basal (green) for W401F/G551D channels. The number above each bar represents the number of patches.

FIGURE 4.

Single-channel kinetics of G551D channels with W401Y,W401F or H1348G mutations. A–D, current traces for optimized G551D channels, recorded in the presence (top) or absence (bottom) of 2.75 mm ATP. E, mean open time for G551D channels and those mutant channels in A–D. Asterisk, p < 0.01. F, estimated increase of the opening rate (R_co) upon the application of ATP for G551D-CFTR and mutant channels in A–D. There is no error bar for this panel as the ratio R_co−ATP/R_co−Basal was estimated by using data from two sets of different experiments. It is noted that some short openings could be observed for optimized G551D channels, implicating that ATP-independent openings may still be present. Therefore, it is possible that our kinetic analysis, by lumping all opening events into one single population, could underestimate the true open time of ATP-induced openings. If so, the subsequent calculation (see “Experimental Procedures”) for the opening rate could overestimate the effect of ATP. Nevertheless, this analytic imprecision does not affect our conclusion that ATP increases the P_o of optimized G551D channels mainly by prolonging the channel open time. The number above each bar represents the number of patches.

FIGURE 5.

Effects of W401F/H1348G mutations on WT and ΔF508 channels. A, 30-s single-channel recordings of WT and W401F/H1348G channels exposed to 2.75 mm ATP. B, current recordings of ΔF508 and ΔF508/W401F/H1348G channels in the presence of 2.75 mm ATP. C–E, mean open time (C), open probability (D), and opening rate (E) extracted from single-channel experiments as shown in A and B. Asterisk, p < 0.01. The number above each bar represents the number of patches.