ABCMdb

PMID: 18167357

Bompadre SG, Li M, Hwang TC
Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog.
J Biol Chem. 2008 Feb 29;283(9):5364-9. Epub 2007 Dec 30., 2008-02-29 [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly551Asp Mechanism of G551D-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) Potentiation by a High Affinity ATP Analog*□S Received for publication,November 16, 2007 Published, JBC Papers in Press,December 30, 2007, DOI 10.1074/jbc.M709417200 Silvia G. Bompadre‡1 , Min Li‡ , and Tzyh-Chang Hwang‡§ From the ‡ Dalton Cardiovascular Research Center and the § Department of Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia, Missouri 65211 Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel gated by ATP binding and hydrolysis at its nucleotide binding domains (NBD). Login to comment 3 ABCC7 p.Gly551Asp The G551D mutation in ABP2, the third most common cystic fibrosis-associated mutation, abolishes ATP-dependent gating, resultinginanopenprobabilitythatisϳ100-foldlowerthanthatof wild-type channels. Login to comment 4 ABCC7 p.Gly551Asp Interestingly, we found that the ATP analog N6 -(2-phenylethyl)-ATP (P-ATP) increases G551D currents mainly by increasing the open time of the channel. Login to comment 6 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Introducingmutationsthatlowerthenucleotidebindingaffinityat ABP2 did not alter significantly the effects of P-ATP on G551D-CFTR, whereas an equivalent mutation at ABP1 (consisting of the Walker A and B motifs of NBD1 and the signature sequence of NBD2) dramatically decreased the potency of P-ATP, indicating that ABP1 is the site where P-ATP binds to increase the activity of G551D-CFTR. Login to comment 16 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The importance of the signature sequence in CFTR gating is attested by the fact that mutations such as G551D and G1349D in thisregionoftheproteinareassociatedwithCF.G551D,locatedin the signature sequence of NBD1, is one of the most common CF-associated mutations (the Cystic Fibrosis Mutation Database). Login to comment 17 ABCC7 p.Gly551Asp G551D-CFTR channels exhibit much lower open probability than wild-type(WT)channels(7-9),andconsequently,thepatientscar- rying this mutation present a severe phenotype (10, 11). Login to comment 18 ABCC7 p.Gly551Asp G551D has been used as a model in pharmacological characterizations of CFTR (9,12-15),butthefundamentalmechanismforthefunctionaldefect has been unclear until recently. Login to comment 19 ABCC7 p.Gly551Asp We found that the G551D mutation completely eliminates the ability of ATP to increase the opening rate of the channel (7), consistent with the idea that ATP binding to ABP2 is critical for the ATP-dependent opening of CFTR channels(6).TheobservedlowactivityofG551D-CFTRlikelyrep- resents the rare ATP-independent openings seen with the WT-CFTR channels in the absence of ATP (7, 16). Login to comment 20 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Although G551D-CFTR does not respond to ATP, interestingly Cai et al. (9) showed that the ATP analog 2Ј-deoxy-ATP increases the Po of G551D-CFTR. Login to comment 21 ABCC7 p.Gly551Asp It remains unclear which nucleotide binding sites this ATP analog acts on to potentiate G551D-CFTR. Login to comment 23 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Since the G551D mutation, located in ABP2, may not affect the function of ABP1, we tested the hypothesis that P-ATP potentiates G551D-CFTR by binding to ABP1. Login to comment 24 ABCC7 p.Gly551Asp We found that P-ATP increases G551D cur- * This work was supported by National Institutes of Health Grants NIHR01DK55835 and NIHR01HL53455 (to T. C. H.) and NIHK01DK075408 (to S. G. Login to comment 38 ABCC7 p.Gly551Asp By introducing mutations that lower the nucleotide binding affinity at each NBD, we were able to conclude that this effect of P-ATP on G551D-CFTR is through binding of the nucleotide to ABP1. Login to comment 40 ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly EXPERIMENTAL PROCEDURES Site-directed Mutagenesis-The constructs containing single mutations (G551D, W401G, and Y1219G) have been described previously (6, 7). Login to comment 41 ABCC7 p.Gly551Asp Additional point mutations were introduced into G551D-CFTR by using a QuikChange XL kit (Stratagene, La Jolla, CA) according to the manufacturer`s protocol. Login to comment 55 ABCC7 p.Gly551Asp Since the activity of G551D is extremely low, the number of channels in the membrane patch cannot be ascertained. Login to comment 57 ABCC7 p.Gly551Asp To better estimate the mean closed timeweemployedastrategybasedonourpreviousresults(7).Wefirst obtained␶o inthecontrolconditionasdescribedabove.SincethePo of G551D-CFTRisϳ0.004(i.e.1/120 of the Po for WT-CFTR), ␶c can be calculated based on the equation, ␶c ϭ (␶o/Po) - ␶o. Login to comment 62 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp RESULTS Effect of P-ATP on G551D-CFTR-Fig. 1A shows a continuous current trace from an excised inside-out patch of a Chinese hamster ovary cell expressing G551D-CFTR. Login to comment 64 ABCC7 p.Gly551Asp Reversible activation of G551D-CFTR by P-ATP. Login to comment 65 ABCC7 p.Gly551Asp A, P-ATP, a high affinity ATP analog, increases G551D-CFTR activity. Login to comment 68 ABCC7 p.Gly551Asp C, P-ATP dose-response relationship for G551D-CFTR channels. Login to comment 71 ABCC7 p.Gly551Asp ATP Analog Potentiates G551D-CFTR FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5365 atUniversityofNorthCarolinaatChapelHill,onAugust,2011www.jbc.orgDownloadedfrom protein kinase (not shown). Login to comment 74 ABCC7 p.Gly551Asp Similar results were obtained for G551D-CFTR expressed in human epithelial cells CFPAC-1 (see supplemental materials). Login to comment 75 ABCC7 p.Gly551Asp To further quantify the effect of P-ATP on G551D currents, we measured the channel activity at different [P-ATP] and calculated the ratio between the mean current in the presence of P-ATP and the mean control current (washout). Login to comment 76 ABCC7 p.Gly551Asp The P-ATP dose-response relationship (Fig. 1C) shows that G551D-CFTR currents can be enhanced as much as 7-fold at 50 ␮M P-ATP with a K1/2 of ϳ6 ␮M. In patches with sporadic openings, we could observe in detail the effect of P-ATP on the channels. Login to comment 83 ABCC7 p.Gly551Asp The results indicate that the increase of the G551D channel activity by P-ATP is achieved mainly by an increase in the open time of the channel. Login to comment 84 ABCC7 p.Gly551Asp G551D channels have extremely long closed times, ␶o ϽϽ ␶c; consequently, the channel activity is directly proportional to the open time (i.e. Po ϭ ␶o/(␶o ϩ ␶c) Х ␶o/␶c). Login to comment 86 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Based on these results, we can safely conclude that the increase in G551D-CFTR activity in the presence of P-ATP is mainly due to an increase in ␶o. ATP and P-ATP Compete for the Same Biding Sites-We next tested whether the effect of P-ATP on G551D-CFTR is through binding to the same site(s) ATP may bind. Login to comment 87 ABCC7 p.Gly551Asp Since ATP has little effect on G551D, one expects that the effect of P-ATP will be diminished by the presence of ATP if they share a common binding site. Login to comment 89 ABCC7 p.Gly551Asp In the presence of ATP, the effect of P-ATP is dramatically reduced, but removal of ATP rapidly restores the potentiation of P-ATP on G551D-CFTR, suggesting that these two nucleotides can readily exchange at a binding site. Login to comment 92 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Where Does P-ATP Bind to Increase the G551D-CFTR Current?-To determine to which ABP P-ATP binds to exert its effect on G551D-CFTR channels, we introduced mutations that lower the nucleotide biding affinity at each ABP. Login to comment 93 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly As demonstrated previously, W401G and Y1219G are two mutations that can serve thispurpose.Trp-401wasshowninteractingdirectlywiththeade- nine ring of ATP via a stacking mechanism in the crystal structure of NBD1 from human CFTR (Ref. 19, Protein Data Bank (PDB) code 1XMI). Login to comment 95 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly It was found that, under the WT-CFTR background, the Y1219G mutation, but not the W401G mutation, causes a right- wardshiftoftheATPdose-responsecurve(6).Asimilarresultwas observed for P-ATP (see supplemental materials). Login to comment 96 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly Although the Y1219G mutation decreases the apparent affinity of ATP and P-ATP in WT background, introducing this mutation into the G551D background has little influence on the FIGURE 2. Login to comment 97 ABCC7 p.Gly551Asp P-ATP dependent gating of G551D-CFTR in excised patches. Login to comment 98 ABCC7 p.Gly551Asp A, single-channel current traces for G551D-CFTR in the presence and absence of 10 ␮M P-ATP. Login to comment 99 ABCC7 p.Gly551Asp B, summary of the mean open time and the mean closed time for G551D-CFTR in the presence and absence of P-ATP (n ϭ 11). Login to comment 104 ABCC7 p.Gly551Asp The presence of ATP reduces the potentiation effect of P-ATP, but removal of ATP rapidly restores the effect of P-ATP on G551D-CFTR, indicating that these two nucleotides can readily exchange at a binding site (n ϭ 6). Login to comment 105 ABCC7 p.Gly551Asp ATP Analog Potentiates G551D-CFTR 5366 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER •FEBRUARY effect of P-ATP. Login to comment 106 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly Fig. 4A shows a representative trace of G551D/ Y1219G-CFTR channels in the absence and presence of 10 ␮M P-ATP. Login to comment 107 ABCC7 p.Gly551Asp 10 ␮M P-ATP increased the activity of this double mutant by 5.0 Ϯ 0.6-fold (n ϭ 7), and as in the case of G551D channels, this increase in activity is mainly due to an increase in the open time of the channel (Fig. 4B). Login to comment 109 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly The G551D/Y1219G-CFTR P-ATP dose response is very similar to the G551D-CFTR dose response, suggesting that lowering the binding affinity at the ABP2 site does not alter the effect of P-ATP. Login to comment 110 ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly In contrast, introducing W401G in the G551D background significantly reduced the effect of P-ATP. Login to comment 111 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly The activity of W401G/G551D-CFTR channels in the presence of 10 ␮M P-ATP is smaller than the activity of G551D channels under the same conditions. Login to comment 112 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly The mean current -fold increase is 1.9 Ϯ 0.2 for W401G/G551D-CFTR when compared with 6.2 Ϯ 0.7 for G551D-CFTR. Login to comment 115 ABCC7 p.Trp401Gly The rightward shift of the P-ATP dose-response relationship suggests that mutating Trp-401 to glycine at ABP1 lowers the P-ATP binding affinity (Fig. 6). Login to comment 116 ABCC7 p.Gly551Asp We thus conclude that P-ATP binds to ABP1 to increase the open time of G551D channels and consequently enhance the activity of this mutant. Login to comment 117 ABCC7 p.Gly551Asp DISCUSSION The current results show that P-ATP, a high affinity ATP analog, binds to ABP1 to enhance G551D-CFTR, and it does so mainly by increasing the open time of the channel. Login to comment 119 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Mechanistically, it establishes that nucleotide binding at ABP1 can stabilize the open state even for G551D-CFTR, a mutant irresponsive to ATP; pharmacologically, it suggests that ABP1 may be a potential molecular target for drugs that could potentiate G551D-CFTR. Login to comment 125 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Previously, we showed that ATP fails to increase the opening rate of G551D-CFTR (7), and this observation led us to conclude that G551D-CFTR presents only ATP independent openings. Login to comment 127 ABCC7 p.Gly551Asp These results are consistent with the ideas that ABP2 is the site that couples ATP binding to the channel opening and that the G551D mutation specifically abolishes the function of ABP2. Login to comment 128 ABCC7 p.Gly551Asp Thus, other nucleotides such ADP or AMP-PNP, which presumably act on ABP2 (7, 16), were not effective on G551D-CFTR (7). Login to comment 130 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly P-ATP effect on G551D/Y1219G-CFTR. Login to comment 131 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly A, single-channel traces of Y1219G/G551D-CFTR in the presence or absence of P-ATP. Login to comment 132 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly The mutation Y1219G (located in ABP2) decreases the apparent P-ATP binding affinity under the WT background (see supplemental materials), but when introduced into the G551D background, the P-ATP potentiation is not affected. Login to comment 133 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly B, comparison of the mean currents and mean open times for G551D and G551D/Y1219G in the presence of 10 ␮M P-ATP (n ϭ 5-11). Login to comment 135 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly P-ATP dose-response relationships for G551D/Y1219G-(Œ) and W401G/G551D-CFTR (f). Login to comment 138 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly The K1/2 values are 13 Ϯ 5 and 79 Ϯ 30 ␮M for G551D/ Y1219G-CFTR and W401G/G551D-CFTR, respectively. Login to comment 139 ABCC7 p.Gly551Asp The dashed line represents the P-ATP dose-response relationship for G551D-CFTR shown in Fig. 1. Login to comment 140 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ATP Analog Potentiates G551D-CFTR FEBRUARY 29, 2008•VOLUME 283•NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 5367 that for G551D-CFTR, the function of ABP1 may not be jeopardized since P-ATP can bind to this site to stabilize the open channel conformation. Login to comment 141 ABCC7 p.Gly551Asp The significance of this conclusion is that the G551D construct presents a great opportunity to investigate specifically the interaction of nucleotides with ABP1, a yet challenging issue for several reasons. Login to comment 145 ABCC7 p.Gly551Asp It is interesting to note that the effects of P-ATP echo what has been reported by Cai et al. (9), that 2Ј-deoxy-ATP increased the activity of G551D channels. Login to comment 148 ABCC7 p.Gly551Asp However, it should be pointed out that it is extremely difficult to accurately estimate ␶c because of the low Po of G551D. Login to comment 150 ABCC7 p.Gly551Asp Given the extremely low Po of G551D, this practice inevitably results in an underestimation of the closed time. Login to comment 152 ABCC7 p.Gly551Asp If it turns out that 2Ј-deoxy-ATP also binds to ABP1 to potentiate G551D-CFTR, it may be possible to design even more effective nucleotides for this mutant. Login to comment 153 ABCC7 p.Gly551Asp If we assume that the ATP-independent openings of G551D involve the dimerization of the NBDs, an outstanding question remains to be answered. Login to comment 154 ABCC7 p.Gly551Asp When G551D channels open, is ABP2 occupied or empty? Login to comment 156 ABCC7 p.Gly551Asp Since the G551D mutation is located in the signature sequence of NBD1 (ABP2), one could assume that the nucleotide binding at ABP2 is not affected by this mutation, but instead, the post-binding events may be impaired. Login to comment 160 ABCC7 p.Gly551Asp Alternatively, if an NBD dimer can form in the presence of ATP (or P-ATP) bound at ABP2, we need to explain why ATP (or P-ATP) fails to increase the opening rate of G551D channels. Login to comment 162 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Since we do not know whether the G551D open state involves the dimerization of the NBDs, we need to consider the possibility that the openings of G551D may be unrelated to the dimerization of the NBDs. Login to comment 164 ABCC7 p.Gly551Asp Thus, we cannot rule out the possibility that the openings of G551D are decoupled from the NBD dimerization. Login to comment 165 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp However, since binding of P-ATP to ABP1 decreases the closing rate of the G551D channels, we have to conclude that even under this scenario, the transmembrane domains are not totally decoupled from the NBDs in G551D-CFTR. Login to comment 168 ABCC7 p.Gly551Asp G551D is a disease-associated mutation that traffics normally to the membrane (7, 25) but with defective gating. Login to comment 170 ABCC7 p.Gly551Asp Many compounds have been shown to increase G551D-CFTR currents (9, 12, 13, 15, 26), but none of them seems to be potent enough to completely restore the Po of the channel to WT levels. Login to comment 172 ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly P-ATP effect on W401G/G551D-CFTR. Login to comment 173 ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly A, single-channel traces of W401G/G551D-CFTR in the presence or absence of P-ATP. Login to comment 175 ABCC7 p.Gly551Asp ATP Analog Potentiates G551D-CFTR 5368 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 283•NUMBER 9•FEBRUARY understanding of how these compounds work or where they bind, it is very difficult to design a drug that may accomplish the task of rescuing the defective channel. Login to comment 178 ABCC7 p.Gly551Asp Our observation that P-ATP potentiates G551D-CFTR by binding to ABP1 suggests that ABP1 may be a target for drugs to bind and enhance the mutant channel activity. Login to comment 181 ABCC7 p.Gly551Asp It should be interesting to examine biochemically whether the G551D mutation abolishes nucleotide occlusion. Login to comment 182 ABCC7 p.Gly551Asp Nevertheless, we believe that these new findings will likely serve as a structural framework for the future development of potentiators for CFTR mutants such as G551D. Login to comment