ABCMdb

PMID: 17353351

Bompadre SG, Sohma Y, Li M, Hwang TC
G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects.
J Gen Physiol. 2007 Apr;129(4):285-98. Epub 2007 Mar 12., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp (c) The Rockefeller University Press $15.00 Volume 129 Number 4 April 2007 285-298 http://www.jgp.org/cgi/doi/10.1085/jgp.200609667 285 A RT I C L E G551D and G1349D, Two CF-associated Mutations in the Signature Sequences of CFTR, Exhibit Distinct Gating Defects Silvia G. Bompadre,1,2 Yoshiro Sohma,2,3 Min Li,1,2 and Tzyh-Chang Hwang1,2 1Department of Medical Pharmacology and Physiology, 2Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211 3Department of Physiology, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). Login to comment 3 ABCC7 p.Gly551Asp G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Login to comment 4 ABCC7 p.Gly551Asp Patients carrying the G551D mutation present a severe clinical phenotype. Login to comment 5 ABCC7 p.Gly1349Asp On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Login to comment 9 ABCC7 p.Gly551Asp The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is ‫-001ف‬fold smaller than WT-CFTR. Login to comment 10 ABCC7 p.Gly551Asp G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg2+]. Login to comment 11 ABCC7 p.Gly551Asp The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. Login to comment 12 ABCC7 p.Gly1349Asp G1349D-CFTR maintains ATP dependence, albeit with a Po ‫-01ف‬fold lower than WT. Login to comment 13 ABCC7 p.Gly1349Asp Interestingly, compared to WT results, the ATP dose-response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. Login to comment 14 ABCC7 p.Gly1349Asp G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Login to comment 25 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp These mutations can be divided into four classes based on the mechanisms that disrupt CFTR function (Welsh and Smith, 1993): defective protein production (I); defective protein processing (II); defective activation and regulation (III), including G551D and G1349D; and defective conductance (IV). Login to comment 26 ABCC7 p.Gly551Asp G551D is the third overall most common CF mutation with a worldwide frequency of ‫%3ف‬ (www.genet .sickkids.on.ca/cftr). Login to comment 28 ABCC7 p.Gly551Asp The result of this glycine-to-aspartate mutation at position 551 is a significantly decreased Correspondence to Tzyh-Chang Hwang: hwangt@health.missouri.edu Abbreviations used in this paper: ABC, ATP-binding cassette; ABP, ATP binding pocket; CFTR, cystic fibrosis transmembrane conductance regulator; NBD, nucleotide binding domain. Login to comment 32 ABCC7 p.Gly1349Asp However, the G1349D mutation is associated with a milder clinical phenotype (Brancolini et al., 1995, Salvatore et al., 2002). Login to comment 33 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Although G551D-CFTR and G1349D-CFTR have been used in pharmacological studies for years (e.g., Illek et al., 1999; Galietta et al., 2001; Moran et al., 2005; Pedemonte et al., 2005), little is known about the mechanism responsible for their dysfunction due to limited functional studies of these two mutants at a single-channel level (e.g., Cai et al., 2006). Login to comment 36 ABCC7 p.Gly551Asp Using a fluorescence-based assay they observed that G551D-CFTR channels lack functional activity, even though the channels can be normally phosphorylated in the R domain (Chang et al., 1993). Login to comment 37 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Logan et al. (1994) reported that as a consequence of the G551D, or G1349D mutation, nucleotide binding to isolated recombinant NBD1 and NBD2 is decreased. Login to comment 39 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Wilkinson et al. (1996) measured the rates of activation and deactivation of macroscopic CFTR currents in Xenopus oocytes and found that the apparent on rate of channel activation by cAMP for G551D-CFTR or G1349D-CFTR was drastically reduced. Login to comment 42 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp We have studied the response of G551D-CFTR and G1349D-CFTR to ATP, ADP, and AMP-PNP in excised inside-out membrane patches. Login to comment 44 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The G551D mutation completely abolishes the response of the channel to ATP and ADP, whereas the G1349D mutation remains responsive to ATP and the ATP-induced activity can be inhibited by ADP. Login to comment 46 ABCC7 p.Gly1349Asp Interestingly, compared with WT data, the ATP dose-response relationship for G1349D-CFTR is less steep and shows an increase of the apparent affinity for ATP. Login to comment 47 ABCC7 p.Gly1349Asp These features of the ATP dose response for G1349D-CFTR could be reproduced by assuming that ATP binding to ABP1, where the mutation is located, hinders channel opening. Login to comment 50 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Point mutations (G551D and G1349D) were introduced into WT-CFTR by QuikChange XL method (Stratagene). Login to comment 60 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Thus the G551D mutation is located in ABP2 and the G1349D mutation is located in ABP1. Login to comment 102 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp AMP-PNP was purchased from Roche and stored as 250 mM stock in H2O at -20°C. R E S U LT S Expression of G551D-CFTR and G1349D-CFTR It has been previously reported that neither G551D-CFTR nor G1349D-CFTR exhibit trafficking defects in COS cells (Gregory et al., 1991). Login to comment 105 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp G551D-CFTR and G1349D-CFTR expression in CHO cells. Login to comment 106 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp (A) Western blot analysis for WT-, G551D-, and G1349D-CFTR. Login to comment 110 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp (B) Mean current densities obtained from whole-cell experiments for G551D (n = 30), G1349D (n = 29), and WT (n = 20). Login to comment 111 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Columns and error bars indicate means ± SEM, * indicates P < 0.01 between G551D and G1349D. Login to comment 115 ABCC7 p.Gly551Asp We transfected CHO cells with WT-CFTR, G551D-CFTR, and G1349-CFTR, in parallel, using the same amount of DNA. Login to comment 119 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Fig. 2 B shows that the current densities of G551D (n = 30) and G1349D (n = 29) mutants are lower than that of WT-CFTR (n = 20). Login to comment 120 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp However, the current density of G1349D-CFTR is significantly larger than that of G551D-CFTR (P < 0.01), suggesting that the Po of G1349D-CFTR is higher than that of G551D-CFTR. Login to comment 121 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ATP-dependent Gating of G551D-CFTR and G1349D-CFTR To investigate the mechanism responsible for the different gating behavior of G551D and G1349D mutants, we studied both mutants in excised inside-out membrane patches. Login to comment 125 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Gating of G551D-CFTR and G1349D-CFTR in excised inside-out membrane patches. Login to comment 126 ABCC7 p.Gly551Asp (A) A recording of G551D-CFTR channel currents in an excised inside-out membrane patch. Login to comment 128 ABCC7 p.Gly551Asp Expanded current traces show the G551D-CFTR channel activity in the presence of ATP + PKA and during washout. Login to comment 129 ABCC7 p.Gly551Asp (B) Repeated addition and removal of ATP in the same patch did not result in significant changes of the channel activity of G551D-CFTR. Login to comment 130 ABCC7 p.Gly1349Asp (C) A recording of G1349D-CFTR channel currents in an excised inside-out membrane patch. Login to comment 135 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp (E) Comparisons of mean macroscopic current amplitude for G551D- (n = 16), G1349D- (n = 18), and WT-CFTR (n = 13). Login to comment 137 ABCC7 p.Gly551Asp In the case of G551D-CFTR, the elicited currents were usually small compared with the macroscopic current seen with WT-CFTR (Fig. 3, compare A and D). Login to comment 138 ABCC7 p.Gly551Asp Surprisingly, when ATP and PKA were removed, the G551D-CFTR current remained unchanged for tens of minutes (n = 46). Login to comment 139 ABCC7 p.Gly551Asp Fig. 3 A shows a 25-min continuous recording of G551D-CFTR. Login to comment 140 ABCC7 p.Gly551Asp We are confident that the observed current arises from G551D-CFTR channels because no activity was observed in nontransfected cells and the observed currents were PKA dependent. Login to comment 141 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The single-channel amplitude of G551D-CFTR is similar to that of WT channels (see below), and furthermore, glibenclamide, a CFTR blocker (Zhou et al., 2002), blocks G551D-CFTR currents to the same extent as it does to WT-CFTR (unpublished data). Login to comment 142 ABCC7 p.Gly551Asp Repeated addition and removal of ATP in the same patch did not result in significant changes of the channel activity (Fig. 3 B), suggesting that G551D-CFTR channel activity is ATP independent. Login to comment 144 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp When the same experiment was performed with G1349D-CFTR, we observed generally larger currents than that of G551D-CFTR (e.g., Fig. 3 C). Login to comment 145 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In addition, unlike G551D-CFTR, G1349D-CFTR channel current decreases immediately upon ATP washout, a property shared with the WT channels (Fig. 3 D). Login to comment 146 ABCC7 p.Gly1349Asp Note that for both WTand G1349D-CFTR a small but significant fraction of the current remained for minutes after nucleotide removal. Login to comment 148 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The steady-state mean current amplitude (a product of the number of functional channels, Po and single-channel amplitude) shows a similar pattern as that of whole-cell recordings (i.e., WT > G1349D > G551D). Login to comment 153 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Since the Po for WT channels in the presence of 1 mM ATP is 0.45 ± 0.04 (see Fig. 9 D), the estimated Po are ≅ 0.0037 for G551D (120 times smaller than the WT Po), and ≅ 0.045 (10 times smaller than the WT Po) for G1349D. Login to comment 155 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Comparison of the mean open times of G551D-, G1349D-, and WT-CFTR. Login to comment 156 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp (A) Expanded single-channel current traces in the presence of 1 mM ATP for G551D-, G1349D-, and WT-CFTR. Login to comment 159 ABCC7 p.Gly551Asp Figure 5. Effect of [Mg2+] in the closing rate of WT and G551D-CFTR. Login to comment 163 ABCC7 p.Gly551Asp (B) G551D-CFTR channel openings remain unaltered when ATP, PKA, and Mg2+ are removed from the perfusion solution (n = 6). Login to comment 168 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp It is interesting to note that Cai et al. (2006) found that the G551D and G1349D mutations shorten the open time of the channels. Login to comment 170 ABCC7 p.Gly1349Asp Since free [Mg2+] is known to affect the open time of WT-CFTR, presumably by altering the ATP hydrolysis rate (Dousmanis et al. 2002), we next examined the effect of Mg2+ on the closing rate of G551Dand G1349D-CFTR channels. Login to comment 171 ABCC7 p.Gly551Asp Fig. 5 shows representative traces for WT and G551D-CFTR. Login to comment 173 ABCC7 p.Gly551Asp That is not the case for G551D-CFTR, where the current remains unaltered when we switch from Mg2+-containing solution to Mg2+- free solution without ATP (n = 6). Login to comment 174 ABCC7 p.Gly551Asp The mean open time for G551D-CFTR in the Mg2+-free solution is 311 ± 85 ms (n = 3), similar to the open time observed in the Mg2+- containing solution. Login to comment 175 ABCC7 p.Gly1349Asp Surprisingly, we also did not observe an increase in the open time in G1349D channels, although some ATP dependence is retained for this mutant (n = 6; unpublished data). Login to comment 176 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Effect of ADP and AMP-PNP on G551D-CFTR and G1349D-CFTR Channels The results shown above indicate that G551D and G1349D, mutations at the equivalent position in the signature sequence of NBD1 and NBD2, respectively, exhibit different gating defects. Login to comment 181 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp However, ADP had little effect on G551D-CFTR currents (n = 10, Fig. 6 B), further confirming that G551D channel currents are ATP independent. Login to comment 182 ABCC7 p.Gly1349Asp In contrast, G1349D-CFTR channels, like WT-CFTR, can be inhibited by ADP; interestingly however, the level of inhibition was slightly but significantly less (P < 0.001), ‫04ف‬ ± 3% (n = 8, Fig. 6 C). Login to comment 185 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In excised patches, after activation with 1 mM ATP + PKA, we applied 2 mM AMP-PNP + 1 mM ATP to WT (Fig. 7 A), G551D (Fig. 7 B), Figure 6. Effect of ADP on G551D, G1349D, and WT-CFTR. Login to comment 186 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Representative current traces for WT-CFTR (A), G551D-CFTR (B), and G1349D-CFTR (C). Login to comment 187 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Currents from both WTand G1349D-CFTR are reduced by ADP, but ADP fails to inhibit G551D-CFTR channel currents. Login to comment 188 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp (D) Summary of percent inhibition by 500 μM ADP for G551D- (n =10), G1349D- (n = 8), and WT-CFTR (n = 8). Login to comment 190 ABCC7 p.Gly1349Asp * indicates P < 0.001 between G1349D and WT. Login to comment 191 ABCC7 p.Gly1349Asp and G1349D channels (Fig. 7 C). Login to comment 196 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In contrast, AMP-PNP failed to increase either the G551D- or G1349D-CFTR currents (Fig. 7, B and C). Login to comment 197 ABCC7 p.Gly1349Asp Upon removal of the nucleotides, most of the G1349D-CFTR channel currents decreased rapidly (Fig. 7 C). Login to comment 199 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp [ATP] Dependence of G551Dand G1349D-CFTR Our data suggest that the G551D mutation abolished nucleotide-dependent gating. Login to comment 200 ABCC7 p.Lys1250Ala ABCC7 p.Tyr1219Gly It remains possible that this mutation lowers ATP binding affinity as other mutations in ABP2 (e.g., Y1219G in Zhou et al., 2006; K1250A in Vergani et al., 2003). Login to comment 201 ABCC7 p.Gly551Asp Since the signature sequence itself is not an ATP binding site before the two NBDs dimerize (Locher et al., 2002; Smith et al., 2002; Lewis et al., 2004), it seems unlikely that the G551D mutation will affect ATP dose-response relationship, which reflects ATP affinity of the closed state when the NBDs are in monomeric configurations. Login to comment 202 ABCC7 p.Gly551Asp G551D-CFTR activity was examined at different [ATP]. Login to comment 203 ABCC7 p.Gly551Asp Fig. 8 A shows that the channel activity of G551D-CFTR is not altered even in the presence of 10 mM ATP. Login to comment 206 ABCC7 p.Gly1349Asp We next examined the ATP dependence of G1349D-CFTR more thoroughly by measuring the activity of the channel at different [ATP]. Login to comment 207 ABCC7 p.Gly1349Asp In excised patches, G1349D-CFTR channels were exposed to 2.75 mM ATP + PKA, and then to various ATP concentrations (from 2 μM to 10 mM). Login to comment 208 ABCC7 p.Gly1349Asp The experiments were performed by quantifying G1349D-CFTR channel current at a test [ATP] that was bracketed with 2.75 mM ATP to ensure the absence of a time-dependent rundown (for details see Zeltwanger et al., 1999). Login to comment 209 ABCC7 p.Gly1349Asp Fig. 9 (A and B) shows representative traces for WTand G1349D-CFTR. Login to comment 210 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Interestingly, the magnitude of current elicited by 50 μM ATP, relative to that with 2.75 mM ATP, is larger for G1349D-CFTR (Fig. 9 B) than for WT-CFTR (Fig. 9 A), suggesting that G1349D-CFTR channels are "more sensitive" to ATP than WT-CFTR. Login to comment 211 ABCC7 p.Gly1349Asp Fig. 9 C shows the normalized ATP dose response for G1349D-CFTR with an overlaid WT-CFTR data for comparison (from Zhou et al., 2006). Login to comment 212 ABCC7 p.Gly1349Asp Interestingly, similar to that of WT-CFTR, the G1349D-CFTR channel activity is saturated at millimolar [ATP], but the ATP dose-response relationship is shifted to the left. Login to comment 215 ABCC7 p.Gly1349Asp Therefore, the difference in the dose response between WTand G1349D-CFTR (apparent Kd and maximal Po) reflects effects of the mutation on the opening rate of the channel. Login to comment 216 ABCC7 p.Gly1349Asp More interestingly, the dose-response curve of G1349D-CFTR is not as steep as that of WT-CFTR. Login to comment 217 ABCC7 p.Gly1349Asp At low micromolar [ATP], the relative fraction of G1349D current is higher than that of WT. Login to comment 218 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Qualitatively speaking, as the [ATP] is increased, the activity of the G1349D mutant does not increase as much as that of WT channels, as if the increasing activation of G1349D-CFTR upon Figure 7. Effect of AMP-PNP on G551D, G1349D, and WT-CFTR. Login to comment 219 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Representative current traces for WT-CFTR (A), G551D-CFTR (B), and G1349D-CFTR (C). Login to comment 221 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Neither G551D- nor G1349D-CFTR responds to AMP-PNP (n = 4 each). Login to comment 223 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp Kinetic Modeling for G1349D-CFTR To explain the unusual ATP dose-response relationship of G1349D-CFTR, we resort to CFTR gating schemes that incorporate two ATP binding sites. Login to comment 231 ABCC7 p.Gly1349Asp We then tried to fit the G1349D data using this scheme. Login to comment 232 ABCC7 p.Gly1349Asp To fit the G1349D data we need to be able to reproduce the shift of the curve, the flattening of the slope, as well as an ‫-01ف‬fold reduced maximal Po. Login to comment 233 ABCC7 p.Gly1349Asp Since the open time for G1349D-CFTR is not significantly different from that of WT channels, kc(hydro) remains unchanged. Login to comment 238 ABCC7 p.Gly551Asp ATP dose-response relationship for G551D-CFTR. Login to comment 239 ABCC7 p.Gly551Asp (A) Representative trace for G551D-CFTR in the presence of 10 mM ATP. Login to comment 241 ABCC7 p.Gly551Asp (B) Normalized ATP dose-response relationship for G551D-CFTR. Login to comment 244 ABCC7 p.Gly1349Asp ATP dose-response for G1349D-CFTR. Login to comment 245 ABCC7 p.Gly1349Asp Representative traces of WT- (A) and G1349D-CFTR (B) channels in response to 50 μM and 2.75 mM ATP. Login to comment 246 ABCC7 p.Gly1349Asp 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. Login to comment 247 ABCC7 p.Gly1349Asp (C) Normalized ATP dose-response relationship for G1349D-CFTR (red circles) and WT-CFTR (blue squares, from Zhou et al., 2006). Login to comment 248 ABCC7 p.Gly1349Asp (D) Relationships between [ATP] and single-channel Po (blue squares for WT, from Zhou et al., 2006, and red open circles for G1349D). Login to comment 249 ABCC7 p.Gly1349Asp The G1349D Po was calculated under the assumption that the maximal Po is 10 times lower than WT Po. Login to comment 257 ABCC7 p.Gly551Asp The parameters for the spontaneous openings were obtained from Bompadre et al. (2005b) (measured for ∆R-CFTR), and we assumed that ATP binding to ABP1 has little effect on the ATP-independent opening (i.e., kO(SPT0) = kO(SPT1) = kO(SPT)), an assumption supported by Zhou et al. (2006) and the current results with the G551D mutation (see Discussion). Login to comment 258 ABCC7 p.Gly551Asp We also assumed that the closing rate of both types of spontaneous openings is the same (kc(SPT0) = kc(SPT1) = kc(SPT)) since the mean open time of G551D-CFTR is approximately the same as the mean open time of WT channels in the absence of ATP. Login to comment 259 ABCC7 p.Gly1349Asp We calculated the open probability of G1349D-CFTR for this expanded model using the same kinetic parameters listed in Table I with two added new parameters (kc(SPT) = 2 s-1, kO(SPT) = 0.006 s-1; fromt Bompadre et al., 2005b). Login to comment 260 ABCC7 p.Gly1349Asp Adding this ATP-independent component could not replicate the overall shape of the ATP dose response for G1349D-CFTR (unpublished data). Login to comment 261 ABCC7 p.Gly1349Asp We then tested the possibility that the G1349D mutation may affect the opening rate for the spontaneous openings. Login to comment 264 ABCC7 p.Gly1349Asp On the other hand, when kc(SPT) and kO(SPT) were kept the same as those of WT channels, the ratio of the G1349D-CFTR current produced by the ATP-independent openings to the maximal current (0.057 ± 0.010) is very close to what the model predicts (0.06). Login to comment 265 ABCC7 p.Gly1349Asp We should point out that even though we could not simulate the flattening of the dose-response relationship for G1349D-CFTR by modifying the opening rate for the spontaneous openings, Kd2, and the ATP-dependent opening rate (kO(2ATP)), we cannot completely rule out the possibility that some combinations of these parameters could produce a curve similar to our data. Login to comment 274 ABCC7 p.Gly1349Asp (C) Normalized ATP dose response for G1349D-CFTR channels (red circles) and different simulated results obtained with Scheme A. Login to comment 279 ABCC7 p.Gly1349Asp The parameter sets A, B, and C correspond to different attempts to fit the G1349D-CFTR data (green, brown, and red lines in Fig. 10 C, respectively). Login to comment 283 ABCC7 p.Gly1349Asp Using Scheme B, we can also obtain a dose-response relationship that reproduces all the features of the G1349D dose response in at least two different ways (Table II). Login to comment 284 ABCC7 p.Gly1349Asp First, without changing any of the WT binding parameters for ABP1 or ABP2, simply reducing kO (1ATP) to 1 s-1 and kO (2ATP) to 0.16 s-1 can produce data strikingly similar to those of G1349D-CFTR (green line in Fig. 11 C). Login to comment 285 ABCC7 p.Gly1349Asp The dose-response curve of G1349D-CFTR can also be successfully reproduced by slightly decreasing the affinity for ATP at ABP2 (e.g., by increasing Kd2 to 280 μM) and simultaneously lowering the opening rate kO (2ATP) to 0.16 s-1 (red line in Fig. 11 C). Login to comment 286 ABCC7 p.Gly1349Asp These results thus suggest that the G1349D mutation decreases the channel opening rate when ABP1 is occupied by ATP (i.e., kO(2ATP)). Login to comment 288 ABCC7 p.Gly551Asp D I S C U S S I O N The data presented in the current studies strongly suggest that the G551D mutation of NBD1 completely eliminates ATP-dependent gating. Login to comment 289 ABCC7 p.Gly551Asp The residual low activity of G551D-CFTR represents ATP-independent gating events. Login to comment 290 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp On the other hand, G1349D-CFTR exhibits a fairly different behavior than G551D-CFTR; it maintains some ATP dependence, but with a lower Po than WT channels due to a lower opening rate. Login to comment 303 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp (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 TA B L E I I A Summary of the Kinetic Parameters used in Fig. 11 for Scheme B Scheme A WT G1349D (a) G1349D (b) Kd1 (μM) 10 10 10 Kd2 (μM) 130 130 280 kO(1ATP) (s-1) 2.5 1 2.5 kO(2ATP) (s-1) 2.5 0.16 0.16 kC(hydro) (s-1) 3 3 3 kO(SPT) (s-1) 0.006 0.006 0.006 kC(SPT) (s-1) 2 2 2 These sets of parameters correspond to the fits obtained for WT-CFTR (blue line in Fig. 11 B) and G1349D-CFTR (a corresponds to the green line in Fig. 11 C; b corresponds to the red line in Fig. 11 C). Login to comment 311 ABCC7 p.Gly551Asp We showed that G551D channels, though still need to be phosphorylated to be functional, do not respond to ATP at all. Login to comment 312 ABCC7 p.Gly551Asp This lack of response to ATP also explains why the G551D mutant is insensitive to ADP since the main effect of ADP is to competitively inhibit channel opening by ATP (Bompadre et al., 2005a). Login to comment 314 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp If we propose that all the activity observed with the G551D channels represents spontaneous ATP-independent openings, the Po of G551D should be compatible with that of the ATP-independent activity of WT channels. Login to comment 318 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Although it is very difficult, if not impossible, to estimate precisely the Po for G551D from single-channel kinetic analysis, we made a rough estimation by comparing macroscopic current amplitudes between WT and G551D channels if we assume the number of channels in the membrane patches is about the same. Login to comment 319 ABCC7 p.Gly551Asp Fig. 3 E shows that the average mean current of G551D-CFTR in excised patches is ‫021ف‬ times smaller than that of WT channels. Login to comment 320 ABCC7 p.Gly551Asp This result is consistent quantitatively with our hypothesis that G551D-CFTR only exhibits ATP-independent openings. Login to comment 325 ABCC7 p.Gly551Asp Thus, the G551D mutation more likely hampers the conformational changes at ABP2 that facilitate NBD dimerization (i.e., channel opening by ATP). Login to comment 326 ABCC7 p.Gly551Asp On the other hand, if we assume, here, that the G551D mutation does not affect ATP binding at ABP1 (since the mutation is located at ABP2), the failure of ATP to increase the activity of this mutant supports the notion that ATP binding at ABP1 alone indeed does not catalyze channel opening (Zhou et al., 2006; Scheme B, Fig. 11). Login to comment 327 ABCC7 p.Gly551Asp Then, G551D-CFTR openings represent spontaneous openings either with no ATP bound at the ABPs or with ATP bound at ABP1. Login to comment 330 ABCC7 p.Gly551Asp It is worth to note that Cai et al. (2006) reported that 2`-deoxy ATP can potentiate G551D-CFTR channel activity. Login to comment 332 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In contrast to G551D-CFTR, G1349D-CFTR still maintains some ATP dependence, but has a maximal Po ‫-01ف‬fold lower than WT-CFTR. Login to comment 333 ABCC7 p.Gly1349Asp It is interesting to note that the inhibition of G1349D-CFTR channels by ADP is slightly smaller than the inhibition of WT channels. Login to comment 334 ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp To explain this observation we have to realize that G1349D channels also have ATP-independent openings, and because of a much lower Po at maximal [ATP], the fraction of the total current that originates from the ATP-independent openings is relatively higher in G1349D than in WT channels. Login to comment 335 ABCC7 p.Gly1349Asp Since the ATP-independent activity is insensitive to ADP, the portion of the total current that can be inhibited by ADP is relatively smaller in G1349D than WT channels. Login to comment 336 ABCC7 p.Gly1349Asp If binding of ATP to ABP1 is not essential for channel opening as described above, why does the G1349D mutation, located at ABP1, decrease the maximal Po? Login to comment 337 ABCC7 p.Gly1349Asp Since the open time of the mutant channel does not differ significantly from that of WT channels, we can conclude that the defect of G1349D-CFTR resides in a lowered opening rate. Login to comment 339 ABCC7 p.Gly1349Asp The most intriguing feature of G1349D-CFTR is the flattened ATP dose-response curve, suggesting a negative cooperativity between the two ATP binding sites. Login to comment 340 ABCC7 p.Gly1349Asp Based on the results of our modeling, we propose that this negative cooperativity seen with the G1349D mutation arises from a negative effect on channel opening by ATP binding at ABP1. Login to comment 348 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp It is interesting to note that AMP-PNP does not lock open either G551D or G1349D channels. Login to comment 351 ABCC7 p.Gly551Asp In the case of G551D, it is not surprising that we do not see any effects with AMP-PNP because this mutation completely eliminates ATP-dependent gating. Login to comment 352 ABCC7 p.Gly1349Asp It is however puzzling that AMP-PNP is ineffective on G1349D-CFTR. Login to comment 353 ABCC7 p.Gly1349Asp Interestingly, Cai et al. (2006) reported that pyrophosphate, an agent that potentiates CFTR by a similar mechanism as AMP-PNP, also fails to act on G1349D-CFTR. Login to comment 354 ABCC7 p.Gly1349Asp Perhaps, the aspartate side chain of G1349D-CFTR prevents the formation of a stable locked-open conformation. Login to comment 355 ABCC7 p.Gly1349Asp A similar mechanism may also account for the lack of effect on the open time of G1349D-CFTR by removing free Mg2+. Login to comment 356 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp In addition to the mechanistic insights into how CFTR`s two NBDs work concertedly to gate the channel, our observations also provide a quantitative explanation for the different phenotypes exhibited in CF patients carrying the G551D or G1349D mutation. Login to comment 364 ABCC7 p.Gly551Asp Since genistein increases the activity of G551D-CFTR (e.g., Illek et al., 1999) our results suggest that genistein`s action may involve more than a simple effect on ATP-dependent gating as previously proposed (Weinreich, et al., 1997; Wang et al., 1998). Login to comment 365 ABCC7 p.Gly1349Asp Nevertheless, it is interesting to note that 20 μM genistein increases G1349D-CFTR current density 6.8 ± 1.9-fold (n = 14) compared with forskolin alone. Login to comment 366 ABCC7 p.Gly1349Asp With this increase, G1349D current density is ‫%06ف‬ of that of WT, a level likely sufficient to completely restore the physiological function of chloride secretion. Login to comment 367 ABCC7 p.Gly551Asp On the other hand, 20 μM genistein increases G551D current density by 6.5 ± 2.4-fold (n = 19). Login to comment 368 ABCC7 p.Gly1349Asp This may be adequate to reach a level of activity similar to that of G1349D-CFTR but unlikely to completely restore the physiological function of this mutant channel. Login to comment 413 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp 2006. Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel. Login to comment 473 ABCC7 p.Gly551Asp Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Login to comment 507 ABCC7 p.Gly551Asp Phenylglycine and sulfonamide correctors of defective ∆F508 and G551D cystic fibrosis transmembrane conductance regulator chloride-channel gating. Login to comment