ABCMdb

PMID: 16311240

Cai Z, Taddei A, Sheppard DN
Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel.
J Biol Chem. 2006 Jan 27;281(4):1970-7. Epub 2005 Nov 25., 2006-01-27 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Two CF mutants, G551D and G1349D, affect equivalent residues in the highly conserved LSGGQ motifs that are essential components of the ATP-binding sites of CFTR. Login to comment 3 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp To identify small molecules that rescue the gating defects of G551Dand G1349D-CFTR and understand better how these agents work, we used the patch clamp technique to study the effects on G551Dand G1349D-CFTR of phloxine B, pyrophosphate (PPi), and 2؅-deoxy ATP (2؅-dATP), three agents that strongly enhance CFTR channel gating. Login to comment 4 ABCC7 p.Gly551Asp Phloxine B (5 ␮M) potentiated robustly G551D-CFTR Cl-channels by altering both MBD and IBI. Login to comment 5 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp In contrast, phloxine B (5 ␮M) decreased the IBI of G1349D-CFTR, but this effect was insufficient to rescue G1349D-CFTR channel gating. Login to comment 6 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp PPi (5 mM) potentiated weakly G551D-CFTR and was without effect on the G1349D-CFTR Cl-channel. Login to comment 7 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp However, by altering both MBD and IBI, albeit with different efficacies, 2؅-dATP (1 mM) potentiated both G551Dand G1349D-CFTR Cl-channels. Login to comment 9 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp We conclude that G551Dand G1349D-CFTR have distinct pharmacological profiles and speculate that drug therapy for CF is likely to be mutation-specific. Login to comment 26 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp To explore the mutation specificity of CFTR potentiators and to understand better their mechanism of action, the aim of the present study was to investigate the effects of the CFTR potentiators phloxine B, pyrophosphate (PPi), and 2Ј-deoxy-ATP (2Ј- dATP) on the CF mutants G551D and G1349D. Login to comment 28 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Conversely, we selected the CF mutants G551D and G1349D because these mutants profoundly disrupt channel gating by affecting equivalent residues in the LSGGQ motifs of the two ATP-binding sites of CFTR (G551D, site 2, and G1349D, site 1) (4-6; 15-19) and because G551D is a common CF mutation (2). Login to comment 29 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp To quantify the efficacy with which the different CFTR potentiators restore normal channel gating to G551Dand G1349D-CFTR, we employed high resolution single-channel recording and * This work was supported by the Cystic Fibrosis Trust. Login to comment 39 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp MATERIALS AND METHODS Cells and Cell Culture-For this study, we used mouse mammary epithelial cells (C127 cells) stably expressing either wild-type human CFTR or the CF mutant G1349D and Fischer rat thyroid (FRT) epithelial cells stably expressing the CF mutant G551D.3 C127 cells were generous gifts of Professor M. J. Welsh (University of Iowa, Iowa City, IA) and Dr C. R. O`Riordan (Genzyme, Framingham, MA), whereas FRT cells were a generous gift of Drs. L. J. V. Galietta and O. Zegarra-Moran (Istituto Giannina Gaslini, Genoa, Italy). Login to comment 49 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp To determine the relationship between phloxine B concentration and channel activity for G551Dand G1349D-CFTR, we used membrane patches containing multiple active channels. Login to comment 50 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp For all other studies, we used membrane patches containing small numbers of active channels (wild-type and G1349D-CFTR, Յ4; G551D-CFTR, Յ6). Login to comment 54 ABCC7 p.Gly1349Asp Second, we used experimental conditions that robustly potentiate channel activity to determine the number of active channels in a membrane patch. For wild-type and G1349D-CFTR Cl-channels, channel numbers were counted in the presence of ATP (1 mM) and PKA (75 nM) alone. Login to comment 55 ABCC7 p.Gly551Asp In contrast, the presence of the CFTR potentiators phloxine B or 2Ј-dATP were required to determine the number of G551D-CFTR Cl-channels in a membrane patch. Login to comment 56 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Despite our precautions, we cannot exclude the possibility of unobserved G551Dand G1349D-CFTR Cl-channels in membrane patches. Login to comment 57 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Therefore, values of Po for G551Dand G1349D-CFTR might possibly be overestimated. Login to comment 67 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Po was calculated from either open- and closed-times, as described previously (26), or by using the equation: Po ϭ I/͑n ϫ i͒ (Eq. 2) where n represents the number of active channels in the membrane patch. For wild-type CFTR, only membrane patches that contained a single active channel were used for burst analysis, whereas for G551Dand G1349D-CFTRs, we used membrane patches containing no more than four active channels. Login to comment 70 ABCC7 p.Gly1349Asp For example, for G1349D-CFTR, when n ϭ 1, MBD ϭ 34.4 Ϯ 7. Login to comment 73 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp 4 Z. Cai, J.-H. Chen, and D. N. Sheppard, data not shown Rescue of G551D and G1349D-CFTR by CFTR Potentiators JANUARY 27, 2006•VOLUME 281•NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 1971 and IBI í 2,090 Ϯ 481 ms (n ϭ 12) (p Ͼ 0.1 for both sets of data). Login to comment 88 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp RESULTS The Single-channel Activity of Wild-type, G551D-, and G1349D-CFTRs-Before investigating the rescue of the CF mutants G551D and G1349D by CFTR potentiators, we quantified their single-channel activity. Login to comment 89 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Fig. 1A shows representative single-channel recordings of wild-type, G551D-, and G1349D-CFTR. Login to comment 90 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Like other NBD mutants (3), G551Dand G1349D-CFTR were without effect on i but perturbed severely channel gating (Fig. 1, A and B). Login to comment 92 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp In contrast, G551Dand G1349D-CFTR both attenuated the duration of bursts and prolonged dramatically the interburst interval (Fig. 1A). Login to comment 93 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp As a result, the Po of G551Dand G1349D-CFTR were reduced markedly when compared with that of wild-type CFTR (Fig. 1C). Login to comment 95 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Based on analyses of closed-time histograms (wild-type CFTR, ␶c ϭ 14.87 Ϯ 0.51 ms (n ϭ 10); G1349D-CFTR, ␶c ϭ 17.41 Ϯ 1.49 ms (n ϭ 5); p Ͼ 0.05), we used a burst delimiter (␶c) of 15 ms to discriminate inter-and intraburst closures.6 The MBD of G551Dand G1349D-CFTR were similar and both about one-third that of wild-type CFTR (Fig. 1D). Login to comment 96 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp In contrast, the IBI of G551Dand G1349D-CFTR were prolonged strikingly when compared with that of wild-type CFTR (Fig. 1E). Login to comment 97 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Phloxine B Rescues the Gating Defect of G551D-CFTR but Not That of G1349D-CFTR-The fluorescein derivative phloxine B potentiates efficaciously wild-type and ⌬F508-CFTR Cl- currents (11). Login to comment 98 ABCC7 p.Gly551Asp Like its effects on wild-type CFTR (11), phloxine B (0.1-5 ␮M) potentiated greatly G551D-CFTR Cl- currents, whereas phloxine B (10-40 ␮M) inhibited channel activity (Fig. 2, A and B). Login to comment 99 ABCC7 p.Gly551Asp Interestingly, for both wild-type and G551D-CFTR, phloxine B (5 ␮M) potentiated the maximum Cl- cur- rent (Fig. 2B). Login to comment 100 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp However, because under control conditions the activity of G551D-CFTR is much lower than that of wild-type CFTR (Fig. 1), the potentiation of G551D-CFTR by phloxine B exceeded greatly that of wild-type CFTR (Fig. 2B). Login to comment 103 ABCC7 p.Gly551Asp 6 For G551D-CFTR, no membrane patches containing a single active CFTR Cl-channel were obtained. Login to comment 104 ABCC7 p.Gly551Asp However, given the very large difference in the duration of intra-and interburst closures for G551D-CFTR, misclassification errors when defining bursts should be rare. Login to comment 106 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp The single-channel activity of wild-type (WT), G551Dand G1349D-CFTRs. Login to comment 107 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp A, representative single-channel recordings of wild-type, G551D-, and G1349D-CFTRs in excisedinside-outmembranepatchesfromC127cellsexpressingwild-typeandG1349D-CFTRandFRTcellsexpressingG551D-CFTR.Inthisandsubsequentfigures,unlessotherwise indicated, ATP (1 mM) and PKA (75 nM) were continuously present in the intracellular solution, voltage was -50 mV, and there was a large Cl-concentration gradient across the membrane patch (internal [Cl- ] ϭ 147 mM; external [Cl- ] ϭ 10 mM). Login to comment 109 ABCC7 p.Gly1349Asp For wild-type and G1349D-CFTRs, the membrane patches contained one and two active channels, respectively. Login to comment 110 ABCC7 p.Gly551Asp For G551D-CFTR, four active channels were observed when phloxine B (5 ␮M) was added to the intracellular solution (not shown). Login to comment 111 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp B-E, i, Po, MBD, and IBI of wild-type, G551D-, and G1349D-CFTRs. Login to comment 112 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Columns and error bars indicate means ϩ S.E. (wild-type, n ϭ 20 for Po and i, n ϭ 10 for MBD and IBI; G551D, n ϭ 35 for i, n ϭ 10 for Po, MBD, and IBI; G1349D, n ϭ 25 for i, Po, and MBD but n ϭ 5 for IBI). Login to comment 115 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Rescue of G551D and G1349D-CFTR by CFTR Potentiators 1972 tiates G551D-CFTR Cl- currents, we measured i, Po, MBD, and IBI in the absence and presence of the drug. Login to comment 116 ABCC7 p.Gly551Asp Phloxine B (0.1-40 ␮M) caused a concentration-dependent decrease in i equivalent to that observed with wild-type CFTR (Fig. 2C), suggesting that phloxine B does not potentiate G551D-CFTR by increasing current flow through the channel. Login to comment 117 ABCC7 p.Gly551Asp Instead, phloxine B (1-5 ␮M) potentiated G551D-CFTR by enhancing dramatically channel gating, and thus, Po (Fig. 2, A and D, p Ͻ 0.01). Login to comment 119 ABCC7 p.Gly551Asp Importantly, phloxine B (5 ␮M) prolonged the MBD of G551D-CFTR Cl-channels to a length equivalent to that of wild-type CFTR in the absence of drugs (Figs. 1D and 2E; p Ͼ 0.05). Login to comment 120 ABCC7 p.Gly551Asp However, although phloxine B (5 ␮M) decreased the IBI of G551D-CFTR by 62%, it was still over 11-fold longer than that of wild-type CFTR in the absence of the drug (Figs. 1E and 2F; p Ͻ 0.001). Login to comment 121 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Phloxine B (1-20 ␮M) also had biphasic effects on G1349D-CFTR Cl- currents with phloxine B (5 ␮M) potentiating the maximum current (Fig. 3, AandB)(11).Ofnote,themagnitudeofG1349D-CFTRCl- currentpoten- tiated by phloxine B (5 ␮M) was much smaller than that of G551D-CFTR but equivalent to that of wild-type CFTR (Figs. 2B and 3B). Login to comment 122 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Because the single-channel activity of G1349D-CFTR is greatly diminished when com- paredwiththatofwild-typeCFTR(Fig.1),theseresultsindicatethatphlox- ine B fails to rescue the gating defect of G1349D-CFTR. Login to comment 123 ABCC7 p.Gly1349Asp Phloxine B caused a concentration-dependent reduction in i (Fig. 3C) but no change in the number of active G1349D-CFTR Cl-channels (n ϭ 8, Fig. 3A). Login to comment 124 ABCC7 p.Gly1349Asp Interestingly, G1349D-CFTR Cl-channels tended to open more frequently in the presence of phloxine B (Fig. 3A). Login to comment 126 ABCC7 p.Gly1349Asp However, the IBI of G1349D-CFTR in the presence FIGURE 2. Login to comment 127 ABCC7 p.Gly551Asp Phloxine B (PB) potentiates strongly the single-channel activity of G551D-CFTR. Login to comment 128 ABCC7 p.Gly551Asp A, representative recordings show the effects of phloxine B (1 and 5 ␮M) on the activity of G551D-CFTR Cl-channels. Login to comment 129 ABCC7 p.Gly551Asp B, effects of phloxine B concentration on G551D (filled circles and solid line) and wild-type CFTR (open circles and dotted line). Login to comment 130 ABCC7 p.Gly551Asp Data are means Ϯ S.E. (G551D, n ϭ 5-8; wild-type CFTR, n ϭ 4-9) at each point. Login to comment 132 ABCC7 p.Gly551Asp C, effect of phloxine B concentration on i of G551D- (filled circles and solid line) and wild-type CFTR (open circles and dotted line). Login to comment 134 ABCC7 p.Gly551Asp D-F, effects of phloxine B (5 ␮M) on Po, MBD, and IBI of G551D-CFTR Cl-channels. Columns and error bars indicate means ϩ S.E. (n ϭ 7). Login to comment 138 ABCC7 p.Gly1349Asp Phloxine B potentiates weakly the single-channel activity of G1349D-CFTR. Login to comment 139 ABCC7 p.Gly1349Asp A, representative recordings show the effects of phloxine B (1 and 5 ␮M) on the activity of G1349D-CFTR Cl-channels. Login to comment 140 ABCC7 p.Gly1349Asp B, effects of phloxine B concentration on G1349D-CFTR Cl- currents (filled squares and solid line). Login to comment 143 ABCC7 p.Gly1349Asp C, effect of phloxine B concentration on i of G1349D- (filled squares and solid line) and wild-type CFTR (dotted line). Login to comment 145 ABCC7 p.Gly1349Asp D-F, effects of phloxine B (5 ␮M) on Po, MBD, and IBI of G1349D-CFTR Cl-channels. Columns and error bars indicate means ϩ S.E. (n ϭ 7). Login to comment 148 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Rescue of G551D and G1349D-CFTR by CFTR Potentiators JANUARY 27, 2006•VOLUME 281•NUMBER JOURNAL OF BIOLOGICAL CHEMISTRY 1973 of phloxine B (5 ␮M) was over 5-fold longer than that of wild-type CFTR in the absence of drug. Login to comment 149 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Thus, our data demonstrate that phloxine B rescues the gating defect of G551D-CFTR but not that of G1349D-CFTR. Login to comment 150 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Effects of PPi on the Single-channel Activity of G551Dand G1349D-CFTR-The non-hydrolyzable inorganic phosphate analogue, PPi, enhances robustly wild-type CFTR Cl- currents by (i) increasing the rate of channel opening and (ii) decreasing markedly the rate of channel closure (12, 13). Login to comment 151 ABCC7 p.Gly551Asp Fig. 4A demonstrates that PPi (5 mM) augmented the single-channel activity of G551D-CFTR. Login to comment 154 ABCC7 p.Gly551Asp PPi (5 mM) increased, but not significantly, the Po of G551D-CFTR by enhancing slightly MBD and curtailing weakly IBI (Fig. 4, C-E). Login to comment 155 ABCC7 p.Gly1349Asp Like phloxine B (5 ␮M), PPi (5 mM) failed to potentiate the single-channel activity of G1349D-CFTR (Fig. 5). Login to comment 158 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp 2Ј-Deoxy-ATP Activates Both G551Dand G1349D-CFTR Cl-Channels-Because both phloxine B and PPi failed to potentiate G1349D-CFTR, we searched for other agents that might rescue this CF mutant. Login to comment 176 ABCC7 p.Gly551Asp Fig. 7A suggests that 2Ј-dATP (1 mM) enhanced markedly G551D-CFTR channel gating with the result that the number of active channels increased. Login to comment 177 ABCC7 p.Gly551Asp Quantification of the effects of 2Ј-dATP on G551D-CFTR revealed (i) no significant change in i (Fig. 7B), (ii) a 10-fold increase in Po (Fig. 7C), (iii) a 3.5-fold enhancement of MBD (Fig. 7D), and (iv) a 61% decrease in IBI (Fig. 7E). Login to comment 178 ABCC7 p.Gly551Asp Of note, the MBD of G551D-CFTR in the presence of 2Ј-dATP (1 mM) was similar to that of wild-type CFTR in the presence of ATP (1 mM; Figs. 1D and 7D). Login to comment 179 ABCC7 p.Gly551Asp However, the IBI of G551D-CFTR in the presence of 2Ј-dATP (1 mM) was over 17-fold longer than that of wild-type CFTR in the presence of ATP (1 mM; Figs. 1E and 7E). Login to comment 180 ABCC7 p.Gly1349Asp Finally, we tested the effects of 2Ј-dATP on the single-channel activity of G1349D-CFTR.ReplacementofATP(1mM)by2Ј-dATP(1mM)waswith- 7 Comparable results were observed using the C1 7 O 7 C2 kinetic scheme (Ref. 28 and data not shown). Login to comment 182 ABCC7 p.Gly551Asp Effects of PPi on the single-channel activity of G551D-CFTR. Login to comment 183 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp A, representative recordings show the effects of PPi (5 mM) on the activity of G551D-CFTR Cl-channels. B-E, effects of PPi (5 mM) on i, Po, MBD, and IBI of G551D-CFTR. Login to comment 185 ABCC7 p.Gly551Asp The asterisks indicate values that are significantly different from thecontrolvalue(pϽ0.05).IncreasingthePPi concentration to 10 mM failed to enhance further the single-channel activity of G551D-CFTR (nPo: control,0.07Ϯ0.03;PPi (10mM),0.23Ϯ0.10,nϭ6;pϭ 0.7 versus PPi (5 mM) data). Login to comment 187 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Rescue of G551D and G1349D-CFTR by CFTR Potentiators 1974 out effect on i but augmented G1349D-CFTR channel gating, leading to an increase in the number of active channels (Fig. 8). Login to comment 188 ABCC7 p.Gly1349Asp In the presence of 2Ј-dATP (1 mM), the Po and MBD of G1349D-CFTR increased 3.6- and 1.2-fold, respectively (Fig. 8, C and D), while IBI decreased by 54% (Fig. 8E). Login to comment 189 ABCC7 p.Gly1349Asp However, the MBD and IBI of G1349D-CFTR in the presence of 2Ј-dATP (1 mM) were 63% shorter and 2.6-fold longer, respectively, than those of wild-typeCFTRinthepresenceofATP(1mM;Figs.1,DandE,and8,Dand E). Login to comment 190 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp We interpret our data to suggest that 2Ј-dATP potentiates wild-type, G551D-, and G1349D-CFTR channel gating by similar mechanisms but that 2Ј-dATP incompletely restores normal channel gating to either G551D-CFTR or G1349D-CFTR. Login to comment 191 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp DISCUSSION The CF mutants G551D and G1349D affect equivalent residues in the LSGGQ motifs of NBD1 and NBD2. Login to comment 193 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The CFTR potentiators phloxine B and 2Ј-dATP, but not PPi, augment G551D-CFTR channel gating, whereas only 2Ј-dATP enhances that of G1349D-CFTR. Login to comment 194 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Our results demonstrate that G551Dand G1349D-CFTR have distinct pharmacological profiles. Login to comment 195 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Molecular Mechanisms of CFTR Dysfunction in CF Previous studies demonstrated that G551Dand G1349D-CFTR cause a loss of Cl-channel function by disrupting ATP binding, hydrolysis, and thus, channel gating (17, 19, 31). Login to comment 196 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Building on these data, our quantitative analysis of channel gating reveals that these mutants have exceptionally slow opening rates and very fast closing rates when compared with those of wild-typeCFTR.Toexplaintheseverityofthesegatingdefects,weconsider how G551Dand G1349D-CFTR might perturb the ATP-driven NBD dimerization model of CFTR channel gating (5, 20). Login to comment 197 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp The exceptionally slow rates of G551Dand G1349D-CFTR channel opening suggest that these mutants impede ATP binding to sites 1 and 2 with the result that the rate of NBD dimerization is retarded. Login to comment 198 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Moreover, once the NBD dimer forms in G551Dand G1349D-CFTR Cl-channels, it is inherently unstable. Login to comment 212 ABCC7 p.Gly1349Asp Pyrophosphate (5 mM) fails to potentiate the single-channel activity of G1349D-CFTR. Login to comment 213 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp A, representative recordings show the effects of PPi (5 mM) on the activity of G1349D-CFTR Cl-channels. B-E, effects of PPi (5 mM) on i, Po, MBD, and IBI of G1349D-CFTR. Login to comment 215 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Rescue of G551D and G1349D-CFTR by CFTR Potentiators JANUARY 27, 2006•VOLUME 281•NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 1975 reduced. Login to comment 216 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Of note, using a molecular model of the NBD dimer, Moran et al. (15) demonstrated that G551Dand G1349D-CFTR each destabilize ATP binding to sites 1 and 2. Login to comment 220 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Below, we use the model of Vergani et al. (5) to discuss the effects of CFTR potentiators on wild-type, G551D-, and G1349D-CFTR. Login to comment 223 ABCC7 p.Gly551Asp Consistent with this idea, phloxine B prolonged the MBD of G551D-CFTR (present study). Login to comment 224 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp However, phloxine B also decreased the IBI of G551Dand G1349D-CFTR (present study), suggesting that the drug can promote NBD dimer formation for some, but not other, CF mutants (e.g. ⌬F508 (11)). Login to comment 226 ABCC7 p.Gly1349Asp G1349D-CFTR abolished the phloxine B-induced prolongation of channel openings, suggesting that G1349 either directly or indirectly contributes to the binding site for phlox- ineB.However,becausephloxineBdidnotpotentiatetheCFTRCl- chan- nel in the absence of ATP (11), we consider it unlikely that phloxine B interacts directly with site 1. Login to comment 229 ABCC7 p.Gly551Asp 2Ј-dATP potentiates strongly G551D-CFTR Cl-channels. Login to comment 230 ABCC7 p.Gly551Asp A, single-channel recordings of G551D-CFTR in the presence of either ATP (1 mM) or 2Ј-dATP (1 mM). Login to comment 232 ABCC7 p.Gly551Asp B-E, effects of 2Ј-dATP on i, Po, MBD, and IBI of G551D-CFTR, respectively. Login to comment 236 ABCC7 p.Gly1349Asp G1349D-CFTR Cl-channels are potentiated by 2؅-dATP. Login to comment 237 ABCC7 p.Gly1349Asp A, single-channel recordings of G1349D-CFTR in the presence of either ATP (1 mM) or 2Ј-dATP (1 mM). Login to comment 239 ABCC7 p.Gly1349Asp B-E, effects of 2Ј-dATP on i, Po, MBD, and IBI of G1349D-CFTR, respectively. Login to comment 242 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Rescue of G551D and G1349D-CFTR by CFTR Potentiators 1976 the stability of the NBD dimer. Login to comment 246 ABCC7 p.Gly551Asp Consistent with this idea, G551D-CFTR markedly attenuated PPi potentiation of CFTR. Login to comment 247 ABCC7 p.Gly1349Asp Moreover, G1349D-CFTR abolished the potentiation of CFTR Cl- currents by PPi, suggesting that PPi might also bind to site 1 and accelerate channel opening by providing binding energy to drive NBD dimerization. Login to comment 248 ABCC7 p.Gly1349Asp However, because PPi cannot substitute for ATP in supporting CFTR channel gating (13) and because G1349D-CFTR has global effects on NBD dimer function (15), we suggest that the interaction of PPi with site 2 might energize NBD dimerization. Login to comment 254 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp First, among the agents that we tested, only 2Ј-dATP potentiated the gating behavior of both G551Dand G1349D-CFTR. Login to comment 255 ABCC7 p.Gly1349Asp We speculate that the reason why 2Ј-dATP rescued G1349D-CFTR channel gating, whereas phloxine B and PPi did not, is that 2Ј-dATP binds tightly to both sites 1 and 2. Login to comment 256 ABCC7 p.Gly551Asp Second, 2Ј-dATP prolonged the MBD of G551D-CFTR to a magnitude equivalent to that of wild-type CFTR in the presence of ATP. Login to comment 257 ABCC7 p.Gly551Asp However, 2Ј-dATP only attenuated partially the extended IBI of G551D-CFTR. Login to comment 258 ABCC7 p.Gly551Asp (Note that phloxine B had similar effects on G551D-CFTR). Login to comment 259 ABCC7 p.Gly551Asp These data indicate that for G551D-CFTR, the defect in channel closing is easier to rescue than that of channel opening. Login to comment 260 ABCC7 p.Gly551Asp An explanation for the effects of 2Ј-dATP on G551D-CFTR channel gating is provided by the C1 7 C2 7 O model of CFTR channel gating (30). Login to comment 262 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Given that the IBI of G551D-CFTR is 45-fold longer than that of wild-type CFTR (Fig. 1E), the energy barrier opposing the opening of the G551D-CFTR Cl-channel must be considerably greater than that of wild-type CFTR. Login to comment 263 ABCC7 p.Gly551Asp Thus, although 2Ј-dATP and phloxine B might provide sufficient energy to G551D-CFTR to normalize the rate of channel closing, this energy is insufficient to rescue the defect in channel opening. Login to comment