ABCMdb

PMID: 20861014

Tsai MF, Jih KY, Shimizu H, Li M, Hwang TC
Optimization of the degenerated interfacial ATP binding site improves the function of disease-related mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels.
J Biol Chem. 2010 Nov 26;285(48):37663-71. Epub 2010 Sep 22., 2010-11-26 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly551Asp The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Login to comment 4 ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr ABCC7 p.His1348Gly 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). Login to comment 5 ABCC7 p.Gly551Asp Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. Login to comment 17 ABCC7 p.Gly551Asp For example, the G551D mutation at the NBD1 signature motif (site 2) completely eliminates ATP-catalyzed openings of CFTR, presumably by perturbing normal ATP-site 2 interactions (7). Login to comment 21 ABCC7 p.Gly551Asp Thus, we reasoned that rebuilding the degenerated site 1 of CFTR might compensate for the defective site 2 and thereby improve the function of G551D channels. Login to comment 23 ABCC7 p.Gly551Asp We found that a hydrolyzable ATP analogue PATP potentiates G551D channels by binding to the NBD1 head subdomain (13), a component of site 1. Login to comment 33 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Printed in the U.S.A. NOVEMBER 26, 2010•VOLUME 285•NUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 37663 atUniversityofNorthCarolinaatChapelHill,onAugust8,2011www.jbc.orgDownloadedfrom http://www.jbc.org/content/suppl/2010/11/09/285.48.37663.DC1.html http://www.jbc.org/content/suppl/2010/09/22/M110.172817.DC1.html Supplemental Material can be found at: not be optimized for ATP binding, and thus, only high affinity ATP analogues can support G551D-CFTR gating. Login to comment 34 ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr Here, guided by accumulated structural and functional understanding of NBDs, we first identified W401Y,W401F mutations that strengthened ligand binding in the NBD1 head subdomain. Login to comment 35 ABCC7 p.Gly551Asp We demonstrated that these mutations conferred ATP responsiveness for G551D-CFTR and thus significantly improved the function of this sick channel. Login to comment 36 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr The presence of the Asp-551 residue, which disfavors NBD dimer formation, however, raised the question of whether ATP molecule potentiates W401Y,W401F/G551D channels by binding at the NBD interface (i.e. site 1) or interacting only with the monomeric NBD1. Login to comment 37 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.His1348Gly Our data suggested that the former is likely the case as the effects of ATP on W401F/G551D channels can be further enhanced by the H1348G mutation in the NBD2 tail subdomain. Login to comment 38 ABCC7 p.Gly551Asp These results support the idea that optimization of site 1 for ATP binding can compensate for the disabled site 2 for G551D channels. Login to comment 39 ABCC7 p.Gly551Asp Finally, we demonstrated that the same strategy (i.e. optimizing site 1) to improve G551D-CFTR function could be applied to amend at least partially the functional defects of ⌬F508 channels and increase the open probability (Po) for WT channels. Login to comment 54 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.His1348Gly For the W401F/H1348G/G551D channel, the steady-state open probability (Po) in the presence of ATP was estimated by stationary noise analysis of macroscopic currents using the equation ␴2 /i ϭ ͑1 - Po͒I (Eq. 1) where ␴2 is the variance, i is the unitary current amplitude, and I is the amplitude of the steady-state currents. Login to comment 57 ABCC7 p.Gly551Asp It is noted that noise analysis is inappropriate for those G551D-containing channels with even lower Po as the analysis will be more susceptible to the error of measurements (see supplemental text). Login to comment 62 ABCC7 p.Trp401Phe ABCC7 p.His1348Gly This can be readily done for WT and W401F/ H1348G/⌬F508 channels (as seen in Fig. 5). Login to comment 63 ABCC7 p.Gly551Asp However, for ⌬F508 and all G551D-containing channels tested in the current study, the low Po inevitably results in an uncertainty of the channel number in a patch, and thus, only the mean open time can be measured accurately for these mutants. Login to comment 65 ABCC7 p.Gly551Asp The increase of the opening rate upon the application of ATP to G551D-contaning channels (see Fig. 4F) was estimated indirectly. Login to comment 66 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp First, as the mean closed time (␶c, the reciprocal of the opening rate, Rco) is much longer than the mean open time (␶o) for most G551D-containing channels, the Po for these mutants can be approximated as Po ϭ ␶o/␶c ϭ ␶o ϫ Rco (Eq. 2) From here, the -fold increase of the opening rate upon exposing G551D-containing channels to ATP can be estimated as Rco - ATP Rco - Basal ϭ Po - ATP Po - Basal ϫ ␶o - Basal ␶o - ATP (Eq. 3) By calculating the ratio of macroscopic currents in the presence or in the absence of ATP (see Figs. 2B and 3D), we can obtain the average -fold increase of the single-channel Po induced by ATP (i.e. Po - ATP/Po-Basal). Login to comment 70 ABCC7 p.Gly551Asp RESULTS Fig. 1A shows a representative current recording of G551D channels in an inside-out patch. Login to comment 73 ABCC7 p.Gly551Asp Fig. 1A also shows that PATP increased the basal activity of G551D-CFTR by ϳ6-fold. Login to comment 74 ABCC7 p.Gly551Asp Because this effect was diminished when the conserved aromatic residue that stacks against the adenine ring of ATP in NBD1 (Trp-401) but not in NBD2 (Tyr-1219) was mutated (18, 19), we reported previously that PATP potentiates G551D channels by interacting with NBD1 (13). Login to comment 77 ABCC7 p.Gly551Asp What is the factor that determines whether or not a nucleotide ligand can activate G551D-CFTR upon binding to NBD1? Login to comment 78 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp High Affinity Binding of Nucleotides in NBD1 Improves the Function of G551D Channels-As PATP was reported to assume a higher affinity than ATP in CFTR NBDs (14, 15), we hypothesized that high affinity binding in NBD1 is essential for a nucleotide ligand to activate G551D channels. Login to comment 79 ABCC7 p.Gly551Asp If this hypothesis is correct, one would predict that mutations that enhance nucleotide binding in NBD1 will confer ATP-dependent activation as well as a stronger response to PATP for G551D channels. Login to comment 82 ABCC7 p.Trp401Gly This idea is recapitulated in Fig. 1B, which shows that the lock-open duration (i.e. current decay constant calculated from traces in Fig. 1C, see also "Experimental Procedures") of WT channels elicited by ATP and PPi (left) was shortened by the W401G mutation in NBD1 (middle) but was prolonged when PATP, instead of ATP, was used (right). Login to comment 88 ABCC7 p.Trp401Gly ABCC7 p.Trp401Gly ABCC7 p.Trp401Ile ABCC7 p.Trp401Ile As expected, non-conservative substitutions of Trp-401 with Ile or Gly (W401I and W401G), which are unable to stack FIGURE 1. Login to comment 90 ABCC7 p.Gly551Asp A, a macroscopic current recording of G551D channels in response to ATP or PATP as marked. Login to comment 93 ABCC7 p.Trp401Gly ABCC7 p.Trp401Gly P, PATP. B, current traces showing WTor W401G-CFTR channels locked open by PPi with ATP or PATP. Login to comment 96 ABCC7 p.Trp401Gly Red, WT/ATP ϩ PPi; green, W401G/ATP ϩ PPi; light blue, WT/PATP ϩ PPi. Login to comment 100 ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr ABCC7 p.Trp401Tyr Interestingly, we found that phenylalanine (W401F) appeared better than tyrosine (W401Y), which was in turn superior to tryptophan, in stabilizing the lock-open state, suggesting that W401Y and W401F mutations might enhance nucleotide-NBD1 interactions. Login to comment 103 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr We then incorporated W401Y or W401F mutation into G551D channels, intending to help G551D-NBD1 to bind nucleotide more tightly. Login to comment 106 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr It can be seen that W401F, which yielded a more stable lock-open state in the WT background (Fig. 1D), was also more effective than W401Y in conferring ATP-dependent activation of G551D channels. Login to comment 108 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Plotting the lock-open duration of WT-CFTR shown in Fig. 1D with the nucleotide response for G551D-CFTR in Fig. 2B yielded a positive correlation (supplemental Fig. S3), consistent with our hypothesis that high affinity binding in NBD1 is critical for a nucleotide to activate G551D channels. Login to comment 110 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr First, the response to ATP seen in W401Y,W401F/G551D channels could be due to an altered ligand-NBD1 interaction as proposed, or alternatively, is a result of a restored ability for ATP to gate CFTR through binding to site 2. Login to comment 112 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr Moreover, we also found that mutating the Trp-401-equivalent residue in NBD2 (Y1219G), which greatly decreases the NBD2 ATP affinity (19), had little effect on ATP-mediated activation of W401Y,W401F/ G551D channels (supplemental Fig. S4). Login to comment 113 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr Second, as PPi fails to lock open G551D-containing channels (data not shown), presumably due to the negatively charged Asp-551 side chain in site 2, it is desirable to have another parameter in support of the conjecture that W401Y,W401F mutations do tighten nucleotide binding in G551D-NBD1. Login to comment 114 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr One possible approach is to fit the current relaxation traces of W401Y,W401F/G551D channels upon removal of the nucleotide (Fig. 2A, inset) as the resulting time constants could potentially reflect the mean nucleotide dwell time in NBD1 of these channels (see also under "Discussion"). Login to comment 115 ABCC7 p.Gly551Asp Indeed, the data summarized in Fig. 2C suggest that the two Trp-401 mutations do help G551D-NBD1 to bind nucleotides for a longer period. Login to comment 116 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp A comparison of Fig. 2, B and C, reveals that a stronger response of G551D-containing channels to nucleotides is correlated with a slower current decay upon removal of nucleotides (supplemental Fig. S3), thus again corroborating our proposition that the function of G551D channels can be improved when a nucleotide can bind tightly in NBD1. Login to comment 117 ABCC7 p.Gly551Asp Nucleotide-dependent Activity of G551D-containing Channels Requires Closure of the Interface between NBD1 Head and NBD2 Tail-We then sought to answer the following question. Login to comment 118 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr Does the ATP or PATP molecule that potentiates W401Y,W401F/G551D channels bind in the head subdomain of the monomeric NBD1 or NBD1-NBD2 dimer interface (i.e. site 1)? Login to comment 120 ABCC7 p.Gly551Asp ABCC7 p.Ser1347Gly ABCC7 p.Trp401Phe ABCC7 p.Leu1346Gln ABCC7 p.Gly1349Ile Interestingly, we found that non-conservative mutations L1346Q and S1347G (Fig. 3A) and G1349I greatly reduced the nucleotide-dependent activation of W401F/G551D channels. Login to comment 121 ABCC7 p.Gly551Asp ABCC7 p.Ser1347Gly ABCC7 p.Trp401Phe For example, ATP and PATP, which increased the basal activity of W401F/G551D-CFTR by ϳ12-fold (Fig. 3D, blue dashed line) and ϳ30-fold (green dashed line), respectively, led to only ϳ2.5-and ϳ6-fold current increases when the S1347G mutation was present (Fig. 3D). Login to comment 122 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr These results thus suggest that ATP or PATP resides in site 1 and interacts with both NBD1 head and NBD2 tail subdomains to support gating of W401Y,W401F/G551D channels. Login to comment 123 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr The idea that both the head of NBD1 and the tail of NBD2 are involved in ATP-dependent activation of W401Y,W401F/ G551D channels implicates that optimizing the interactions of ATP with the NBD2 tail subdomain may also improve the func- FIGURE 2. Login to comment 124 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr ABCC7 p.Trp401Tyr W401Y and W401F mutations confer ATP-dependent activa- tionofG551Dchannels.A,applicationofATPorPATPsignificantlyincreased currents of W401Y,W401F/G551D channels. Login to comment 125 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe Insets, current relaxation traces recorded after the removal of ATP (blue box) or PATP (green box) for W401F/ G551D-CFTR. Login to comment 126 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp B, the ratio of ATPor PATP-induced current over the basal current for G551D channels with or without Trp-401 mutations. Login to comment 127 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr 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. Login to comment 128 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The number above each bar represents the number of patches. CFTR Function Improved by Optimized Site 1 37666 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 48•NOVEMBER 26, 2010 tion of G551D-CFTR. Login to comment 131 ABCC7 p.Gly551Asp ABCC7 p.His1348Gly We thus converted the His-1348 residue to Gly (H1348G) in the G551D background. Login to comment 133 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.His1348Gly Moreover, the H1348G mutation further improved the function of W401F/G551D channels so that the application of ATP and PATP increased the basal activity by ϳ25- and ϳ75-fold, respectively (Fig. 3, C and D, and supplemental Fig. S5), further supporting the notion that optimizing ATP binding in site 1 enhances the function of G551D channels. Login to comment 134 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Optimized G551D Channels Enter into Longer Open Bursts- We next examined the channel kinetics for those compound G551D-containing mutants. Login to comment 135 ABCC7 p.Gly551Asp Current traces in Fig. 4, A-D, show discernible openings and closings of G551D channels with Trp-401 or His-1348 mutation, recorded in the presence (upper traces) or absence (lower traces) of ATP. Login to comment 138 ABCC7 p.Gly551Asp The role of NBD2 signature motif in mediating ATP response of optimized G551D channels. Login to comment 139 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ser1347Gly ABCC7 p.Trp401Phe ABCC7 p.His1348Gly 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. Login to comment 140 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.His1348Gly C, H1348G enhanced the response of W401F/G551D-CFTR to ATP. Login to comment 141 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe 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. Login to comment 142 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe Dashed lines, the ratio of IATP/IBasal (blue) and IPATP/IBasal (green) for W401F/G551D channels. Login to comment 145 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr ABCC7 p.His1348Gly Single-channel kinetics of G551D channels with W401Y,W401F or H1348G mutations. Login to comment 146 ABCC7 p.Gly551Asp A-D, current traces for optimized G551D channels, recorded in the presence (top) or absence (bottom) of 2.75 mM ATP. Login to comment 147 ABCC7 p.Gly551Asp E, mean open time for G551D channels and those mutant channels in A-D. Asterisk, p Ͻ 0.01. Login to comment 148 ABCC7 p.Gly551Asp F, estimated increase of the opening rate (Rco) 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 Rco-ATP/Rco-Basal was estimated by using data from two sets of different experiments. Login to comment 149 ABCC7 p.Gly551Asp It is noted that some short openings could be observed for optimized G551D channels, implicating that ATP-independent openingsmaystillbepresent.Therefore,itispossiblethatourkineticanalysis, by lumping all opening events into one single population, could underestimate the true open time of ATP-induced openings. Login to comment 151 ABCC7 p.Gly551Asp Nevertheless, this analytic imprecision does not affect our conclusion that ATP increases the Po of optimized G551D channels mainly by prolonging the channel open time. Login to comment 156 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr ABCC7 p.His1348Gly ABCC7 p.His1348Gly W401F and H1348G Mutations Improve the Function of WT and ⌬F508 Channels-To this point, we have demonstrated that optimizing the interactions of ATP with site 1 components, NBD1 head (W401Y and W401F) and NBD2 tail (H1348G), ameliorates the gating defects of G551D channels, which hold a non-functional site 2. Login to comment 160 ABCC7 p.Trp401Phe ABCC7 p.His1348Gly Indeed, when W401F and H1348G mutations were engineered into WT channels (Fig. 5A), the mean open time of WT-CFTR was more than quadrupled (Fig. 5C) with the already high Po (ϳ0.4) nearly doubled (ϳ0.78, Fig. 5D). Login to comment 162 ABCC7 p.Trp401Phe ABCC7 p.His1348Gly In either case, W401F/H1348G mutations did not significantly alter the opening rate (Fig. 5E). Login to comment 163 ABCC7 p.Gly551Asp Thus, the strategy to restore the function of G551D-CFTR by strengthening nucleotide-site 1 interactions also works to augment the activity of WT or ⌬F508 channels. Login to comment 165 ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr ABCC7 p.His1348Gly First, ATP-site 1 interactions of the CFTR channel can be strengthened by introducing mutations in both the head domain of NBD1 (i.e. W401Y,W401F) and the tail domain of NBD2 (i.e. H1348G). Login to comment 166 ABCC7 p.Gly551Asp Second, enhancing ligand-site 1 interactions improves not only the function of G551D-CFTR with a disabled site 2 but also WT and ⌬F508 channels that rely mostly on site 2 for gating control. Login to comment 176 ABCC7 p.Trp401Tyr Here, we reported that ATP binding in CFTR site 1 is not optimized and can be improved by converting Trp-401 to Tyr/Phe and His-1348 back to Gly, the ABC protein consensus. Login to comment 180 ABCC7 p.Trp401Phe ABCC7 p.His1348Gly Effects of W401F/H1348G mutations on WT and ⌬F508 channels. Login to comment 181 ABCC7 p.Trp401Phe ABCC7 p.His1348Gly A, 30-s single-channel recordings of WT and W401F/H1348G channels exposed to 2.75 mM ATP. Login to comment 182 ABCC7 p.Trp401Phe ABCC7 p.His1348Gly B, current recordings of ⌬F508 and ⌬F508/W401F/ H1348G channels in the presence of 2.75 mM ATP. Login to comment 189 ABCC7 p.Trp401Gly ABCC7 p.Ser1347Gly ABCC7 p.Trp401Ile ABCC7 p.Gly1349Ile These mutations include W401G,W401I (Fig. 1, B-D), which eliminate a ring-ring stacking interaction, S1347G (supplemental Fig. S6), which may break a hydrogen bond between ATP and the NBD2 signature motif, and G1349I (supplemental Fig. S6), whose side chain likely protrudes into site 1 and causes a steric clash with ATP (22-24). Login to comment 191 ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr ABCC7 p.His1348Gly It is this correlation between the chemical nature of mutations and the stability of the lock-open state that grants us the confidence that W401Y,W401F and H1348G mutations, which prolonged the lock-open duration of WT-CFTR, indeed tighten ATP binding in site 1. Login to comment 193 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Because G551D-containing channels cannot be locked open by PPi, presumably because PPi cannot bind stably in site 2 with an Asp-551 side chain, we cannot use the same strategy of measuring lock-open duration to gauge the strength of ligand binding in site 1 for G551D-containing channels. Login to comment 194 ABCC7 p.Gly551Asp Instead, we measured the time constant for the current decay of optimized G551D channels upon removal of nucleotide-containing solutions (Fig. 2A) under the assumption that the current decay reflects dissociation of the nucleotide from site 1. Login to comment 195 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.Trp401Tyr The results shown in Fig. 2C did suggest that W401Y,W401F mutations enhance nucleotide binding in G551D site 1, like in the case of WT site 1. Login to comment 197 ABCC7 p.Gly551Asp Possible Mechanism by Which Optimized Site 1 Improves CFTR Function-The most interesting finding in the current study is that the optimized site 1 not only compensates the defective site 2 in supporting G551D channel function but also enhances site 2-controlled gating of WTand ⌬F508-CFTR. Login to comment 198 ABCC7 p.Gly551Asp Before we discuss possible structural mechanisms underlying these results, we first cover some technical issues about quantification of channel activity for optimized G551D-CFTR. Login to comment 199 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.His1348Gly Unlike WT and ⌬F508 channels, whose Po in the presence or absence of W401F/H1348G mutations (Fig. 5) can be measured with reasonable accuracy, the G551D-containing channels exhibit a Po too low to be derived from single-channel analysis. Login to comment 201 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp One simple method is to normalize the Po of optimized G551D mutants in the presence of ATP to the Po of the ATP-independent activity by calculating the ratio IATP/ IBasal (Figs. 2B and 3D), where I is the macroscopic current amplitude for optimized G551D channels. Login to comment 202 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Theoretically, if these G551D-containing mutants have a Po for basal activity similar to that of G551Dand WT-CFTR (i.e. ϳ0.004, Refs. Login to comment 204 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.His1348Gly For instance, that the W401F/H1348G/G551D channel has an IATP/IBasal of ϳ25 indicates that the Po for this mutant is ϳ0.1. Login to comment 205 ABCC7 p.Gly551Asp ABCC7 p.Trp401Phe ABCC7 p.His1348Gly To verify this value, we performed stationary noise analysis for the W401F/ H1348G/G551D mutant, and the resulting Po of 0.09 Ϯ 0.01 (supplemental Fig. S7) did provide some reassurance. Login to comment 206 ABCC7 p.Gly551Asp ABCC7 p.Trp401Tyr Unfortunately, we cannot use this approach to estimate the Po for other compound mutants, such as W401Y/G551D, as the Po values for these channels are likely too small for noise analysis to be accurate (see supplemental text). Login to comment 209 ABCC7 p.Trp401Phe ABCC7 p.His1348Gly We have observed that W401F/H1348G mutations in site 1 prolonged the mean open time of WT-CFTR (Fig. 5C). Login to comment 211 ABCC7 p.Trp401Phe ABCC7 p.His1348Gly Thus, it appears that a stronger ATP binding in site 1, due to the presence of W401F/H1348G mutations, can allosterically tighten the connection between two NBDs around site 2, thereby slowing down channel closure. Login to comment 213 ABCC7 p.Gly551Asp For optimized G551D channels, kinetic analysis showed that in the presence of ATP, these channels exhibited a slow opening rate (Fig. 4F) similar to that of ATP-independent gating but a prolonged open time (Fig. 4E) comparable with that of site 1-optimized WT-CFTR. Login to comment 214 ABCC7 p.Gly551Asp Thus, the restored ATP-dependent activity of these G551D-containing channels is mainly due to an increased stability of the open state upon application of ATP. Login to comment 215 ABCC7 p.Gly551Asp As site 2, which mediates rapid channel opening in WT-CFTR, is disabled by the G551D mutation, the slow opening rate is an expected observation. Login to comment 220 ABCC7 p.Gly551Asp Here, if we take one step further in hypothesizing that the open state for G551D-containing channels, like WT-CFTR, also represents an NBD dimer, we can explain the ATP-elicited long open bursts with the same mechanism used for site 1-optimized WT-CFTR; that is, the tight ATP-site 1 interaction allosterically delays partial separation of NBDs. Login to comment 221 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly However, this NBD dimer state, if it exists, may have an unoccupied site 2 to avoid a possible steric clash between ATP and the Asp-551 residue, an idea resonant with our third conclusion that NBD2 could remain vacant during ATP-dependent gating of optimized G551D channels as the Y1219G mutation, which disrupts ATP binding in NBD2, posed no functional impact on these channels (supplemental Fig. S4). Login to comment 222 ABCC7 p.Gly551Asp It should be noted here that more experiments will be needed to examine this provisional mechanism, and the crucial postulate that even for G551D-CFTR, NBD dimerization constitutes the fundamental mechanism for coupling NBDs and gating of the pore, particularly requires experimental verification. Login to comment 228 ABCC7 p.Gly551Asp We envisage that those compounds that interact with site 1 to potentiate CFTR mutants can be identified by examining whether ATP serves as a competitive inhibitor for their actions on G551D channels. Login to comment 230 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In this regard, VX-770, a potentiator currently in phase III clinical trials in CF patients carrying G551D-CFTR, is of particular interest in that the prolonged open time of G551D-CFTR channels in the presence of VX-770 appears to be reminiscent of the ATP-dependent gating of site 1-optimized G551D channels described in the current study (31). 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