ABCMdb

PMID: 20406820

Miki H, Zhou Z, Li M, Hwang TC, Bompadre SG
Potentiation of disease-associated cystic fibrosis transmembrane conductance regulator mutants by hydrolyzable ATP analogs.
J Biol Chem. 2010 Jun 25;285(26):19967-75. Epub 2010 Apr 20., 2010-06-25 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly551Asp Two of the most common mutations causing a severe phenotype are G551D and ⌬F508. Login to comment 4 ABCC7 p.Gly551Asp Previously we found that the ATP analog N6 -(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by ϳ7-fold. Login to comment 5 ABCC7 p.Gly551Asp Here we show that 2؅-deoxy-ATP (dATP), but not 3؅-deoxy-ATP, increases the activity of G551D-CFTR by ϳ8-fold. Login to comment 7 ABCC7 p.Gly551Asp This new analog enhances G551D current by 36.2 ؎ 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. Login to comment 8 ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly ABCC7 p.Ser1347Gly We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Login to comment 22 ABCC7 p.Gly551Asp More than 1500 disease-associated mutations have been identified, and they can be grouped into at least four classes according to the different mechanisms by which they cause channel dysfunction: defective protein production, defective protein processing (for example ⌬F508), defective activation and regulation (⌬F508, G551D), and defective conductance. Login to comment 23 ABCC7 p.Gly551Asp G551D, the glycine-to-aspartate missense mutation at position 551 in the signature sequence of NBD1 (ABP2), is the third most common CF-associated mutation. Login to comment 37 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Although ATP fails to open G551D channels, the high affinity ATP analog N6 -(2-phenylethyl)-ATP (P-ATP) can enhance the G551D-CFTR activity by ϳ7-fold mainly by increasing the open time (15). Login to comment 38 ABCC7 p.Gly551Asp Interestingly, 2Ј-deoxy-ATP (dATP), an ATP analog with a similar affinity to ATP, has been reported to increase the activity of G551D to a similar extent as P-ATP (16). Login to comment 39 ABCC7 p.Gly551Asp Because the Po of G551D channels is ϳ100-fold smaller than WT, a 7-fold increase of the Po by these ATP analogs is not expected to restore the activity of the mutant channel to normal levels (compare with Ref. 17). Login to comment 48 ABCC7 p.Gly551Asp In this study, we first confirmed the effects of dATP on G551D channels reported previously by Cai et al. (16). Login to comment 50 ABCC7 p.Gly551Asp Interestingly, we found that 3Ј-deoxy-ATP has a much smaller effect on G551D-CFTR currents in comparison to dATP. Login to comment 51 ABCC7 p.Gly551Asp Because the modifications present in P-ATP and dATP are located in different parts of the ATP molecule and likely interact with different amino acids in the binding pocket, we hypothesized that an ATP analog that contains both modifications could have an additive effect on G551D channels, resulting in a significantly higher increase of the activity. Login to comment 55 ABCC7 p.Gly551Asp This remarkable increase in the activity G551D is achieved by P-dATP binding mainly to ABP1. Login to comment 56 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp This result corroborates not only our previous finding that the Po of G551D-CFTR is very small (14) but also suggests that the increase of Po necessary to rescue the defective G551D channel function is indeed achievable. Login to comment 85 ABCC7 p.Gly551Asp For G551D channels we used the maximum number of opening steps in the presence of P-dATP as the number of channels in the patch to determine the -fold increase of the opening rate induced by the ATP analogs (dATP, 3Ј-dATP, and P-dATP). Login to comment 95 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp RESULTS G551D-CFTR Gating by 2Ј- and 3Ј-deoxy-ATP-We transfected CHO cells with cDNA encoding G551D-CFTR, and performed patch-clamp experiments on the transfected cells in the inside-out mode to test the effect of dATP and 3Ј-deoxy-ATP (3-dATP). Login to comment 96 ABCC7 p.Gly551Asp Fig. 1A shows the effect of 1 mM dATP and 1 mM 3-dATP on G551D-CFTR currents pre-activated with ATP plus PKA. Login to comment 98 ABCC7 p.Gly551Asp Application of 1 mM dATP enhanced G551D-CFTR currents by 7.7 Ϯ 0.6-fold (n ϭ 12), and the potentiation effect was readily reversible when dATP was removed. Login to comment 99 ABCC7 p.Gly551Asp In contrast, in the same patch, application of 1 mM 3-dATP increased the currents of G551D-CFTR only by 2.0 Ϯ 0.2-fold (n ϭ 6). Login to comment 103 ABCC7 p.Gly551Asp Interestingly, 3-dATP had very small effect on the open time of G551D-CFTR (1.5 Ϯ 0.3-fold increase, n ϭ 5), whereas the increase in the opening rate (2.2 Ϯ 0.3, n ϭ 5) was similar to the one induced by dATP. Login to comment 105 ABCC7 p.Gly551Asp Thus, the two hydroxyl groups on the ribose ring of ATP play different roles in the observed effects on G551D-CFTR. Login to comment 106 ABCC7 p.Gly551Asp Of note, this result in G551D-CFTR is quite different from those reported by Aleksandrov et al. (35) for WT-CFTR, where they demonstrate little difference between the effect of 2Ј- and 3Ј-deoxy-ATP. Login to comment 107 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Effects of P-dATP on G551D-CFTR-The two ATP analogs (P-ATP and dATP) that significantly increase the activity of G551D-CFTR have different chemical modifications (an extra ring for P-ATP and a hydroxyl group removed for dATP) in different parts of the ATP molecule (adenine ring and ribose, respectively). Login to comment 108 ABCC7 p.Gly551Asp We reasoned that if these two different modifications act independently, an analog that has both modifications (Fig. 2A, an extra ring in the N6 position of the adenine ring and the hydroxyl group in the 2Ј position of the ribose removed) may have a greater effect on G551D-CFTR currents. Login to comment 110 ABCC7 p.Gly551Asp Indeed, Fig. 2B shows that at 50 ␮M P-dATP can enhance the G551D-CFTR current by 36.2 Ϯ 5.4-fold (n ϭ 9). Login to comment 112 ABCC7 p.Gly551Asp Effect of dATP and 3-dATP on G551D-CFTR activity. Login to comment 116 ABCC7 p.Gly551Asp C, dATP dose-response relationship for G551D. Currents were activated with 1 mM ATP plus PKA, and the mean currents at different [dATP] were normalized to the current level in the absence of dATP (washout) (n ϭ 5-12 for each data point). Login to comment 119 ABCC7 p.Gly551Asp The dose-response relationship for P-dATP (Fig. 2D) demonstrates that the apparent affinity for P-dATP is indeed similar to that of P-ATP on G551D (15). Login to comment 121 ABCC7 p.Gly551Asp In patches showing very few sporadic openings, one can see the significantly prolonged openings of G551D-CFTR in the presence of 10 ␮M P-dATP (Fig. 3A). Login to comment 123 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly Structural Nature of the Interaction between P-dATP and G551D-CFTR-To further understand the effect of P-dATP on G551D channels, we made mutations that lower the apparent binding affinity of ATP in each ABP, W401G in ABP1, and Y1219G in ABP2 (2). Login to comment 124 ABCC7 p.Gly551Asp Previously we have shown that P-ATP potentiates G551D-CFTR by binding to ABP1 (15). Login to comment 126 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly The dose-response relationships of P-dATP for G551D/Y1219G-CFTR (Fig. 4C) show that P-dATP increases the current of the G551D/Y1219G mutant to a somewhat similar extent as for G551D channels. Login to comment 127 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly On the other hand, the dose-response relationship of P-dATP for the W401G/G551D mutant (Fig. 4C) shows a significant rightward shift compared with that of G551D-CFTR accompanied with a considerable reduction of the maximal effect of P-dATP. Login to comment 128 ABCC7 p.Gly551Asp These results suggest that P-dATP, like P-ATP, binds mainly to ABP1 to increase the current of G551D-CFTR channels. Login to comment 130 ABCC7 p.Gly551Asp It has been shown that CFTR channels completely missing NBD2 (i.e. ⌬NBD2-CFTR) exhibit ATP-independent openings similar to those observed in G551D channels (32). Login to comment 133 ABCC7 p.Gly551Asp If P-dATP interacts with both sides of ABP1 (head of NBD1 and tail of NBD2) to potentiate the activity of G551D-CFTR, we predict that introducing mutations that diminish the interac- FIGURE 2. Login to comment 134 ABCC7 p.Gly551Asp Effect of P-dATP on G551D-CFTR activity. Login to comment 136 ABCC7 p.Gly551Asp B, 50 ␮M P-dATP can increase G551D-CFTR currents by 36.2 Ϯ 5.4-fold (n ϭ 9). Login to comment 137 ABCC7 p.Gly551Asp C, summary of the maximum -fold increase in G551D-CFTR activity by the ATP analogs P-ATP, dATP, and P-dATP. Login to comment 139 ABCC7 p.Gly551Asp D, P-dATP dose-response relationship for G551D. Currents were activated with 1 mM ATP plus PKA, and the mean currents at different [P-dATP] were normalized to the current level in the absence of P-dATP (washout) (n ϭ 5-17 for each data point). Login to comment 140 ABCC7 p.Gly551Asp FIGURE 3. P-dATP-dependent gating of G551D-CFTR in excised patches. Login to comment 141 ABCC7 p.Gly551Asp A, single-channel current traces for G551D-CFTR in the presence and absence of 10 ␮M P-dATP. Login to comment 142 ABCC7 p.Gly551Asp B, summary of the -fold increase in the mean current (I), the open time (To), and the opening rate (rCO) for G551D-CFTR in the presence or absence of 10 ␮M P-dATP (n ϭ 6). Login to comment 144 ABCC7 p.Gly551Asp In fact, we have already shown that the mutation of amino acid Trp-401 in the head of NBD1 decreases the effect of P-dATP in G551D channels (Fig. 4, B and C). Login to comment 147 ABCC7 p.Ser1347Gly Mutation of the serine at position 1347 to glycine will likely diminish the interaction of the P-dATP molecule with the signature sequence in NBD2. Login to comment 148 ABCC7 p.Gly551Asp ABCC7 p.Ser1347Gly Fig. 4D shows a representative trace for the mutant G551D/S1347G. Login to comment 150 ABCC7 p.Trp401Gly ABCC7 p.Ser1347Gly In fact the mutation S1347G in NBD2 decreases the potentiation effect of P-dATP to a similar extent as the W401G mutation in NBD1 (Fig. 4C). Login to comment 152 ABCC7 p.Gly551Asp Our data indicate that the interaction of the nucleotides with both sides of ABP1 is responsible for the higher activity of G551D channels in the presence of the above mentioned ATP analogs (see "Discussion" for details). Login to comment 158 ABCC7 p.Gly551Asp The novel high affinity ATP analog P-dATP may serve as a tool to accurately determine the Po of ⌬F508-CFTR channels, if it can considerably increase the channel activity, as observed for G551D channels. Login to comment 162 ABCC7 p.Gly551Asp Potential interactions of ATP analogs with the binding pockets of G551D-CFTR. Login to comment 163 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Ser1347Gly Representative current traces of G551D/Y1219G (A), W401G/G551D (B), and G551D/S1347G (D) in the presence of 10 ␮M P-dATP. Login to comment 164 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Ser1347Gly C, P-dATP dose-response relationships for G551D (red, F), W401G/G551D (blue, E), G551D/Y1219G (green, Œ), and G551D/S1347G (black, f). Login to comment 169 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly ABCC7 p.Ser1347Gly F, comparison between the -fold increase in the current for G551D (red), W401G/G551D (blue), and G551D/S1347G (black) channels in the presence of saturating concentrations of dATP, P-ATP, and P-dATP. Login to comment 170 ABCC7 p.Gly551Asp *, p Ͻ 0.001 versus G551D. Login to comment 187 ABCC7 p.Gly551Asp Although P-dATP greatly potentiated ⌬F508 and G551D-CFTR channels, the effect of the analog on the behavior of the channel was different in both mutants. Login to comment 188 ABCC7 p.Gly551Asp In the case of G551D-CFTR, P-dATP increased mainly the open time of the channels. Login to comment 190 ABCC7 p.Gly551Asp By introducing mutations that diminish the nucleotide binding affinity at both ABPs we found that for G551D channels P-dATP bound to ABP1 to increase the channel activity. Login to comment 192 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly Indeed, introducing the mutation W401G (ABP1) or the corresponding mutation Y1219G (ABP2) in ⌬F508-CFTR resulted in a reduction of the effect of 10 ␮M P-dATP (3.6 Ϯ 0.7 and 0.7 Ϯ 0.1 current -fold increase, respectively), suggesting that binding of P-dATP to both ABPs is involved in mediating the effect of P-dATP on ⌬F508-CFTR channels (Fig. 8). Login to comment 193 ABCC7 p.Gly551Asp DISCUSSION In the current report, we show that the channel activity of G551D-CFTR and ⌬F508-CFTR can be increased dramatically by a structurally defined ATP analog. Login to comment 194 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Our result confirms our previous finding that the Po of G551D-CFTR is very small (14), because a 36-fold increase of the activity of G551D-CFTR can be accomplished with P-dATP (and thus reaching one-third of the maximal Po for WT-CFTR). Login to comment 197 ABCC7 p.Gly551Asp Our previous studies showed that the high affinity ATP analog P-ATP enhanced G551D currents by ϳ7-fold (15). Login to comment 198 ABCC7 p.Gly551Asp Cai et al. (16) reported that the ATP analog dATP also increased G551D currents by ϳ10-fold (ϳ8-fold in this study). Login to comment 204 ABCC7 p.Gly551Asp The newly synthesized analog, combining the modifications of P-ATP (an extra ring in the adenine ring) and dATP (a hydroxyl group missing at the ribose) increases the activity of G551D-CFTR channels by ϳ36-fold and of ⌬F508-CFTR by ϳ20-fold. Login to comment 211 ABCC7 p.Gly551Asp 3 and 6, the main effect of P-dATP on G551D channels is to increase the open time of the channels, whereas for ⌬F508 channels it is the increase in the opening rate. Login to comment 214 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Effect of P-dATP on G551D-CFTR-We have previously shown that the G551D mutation abolishes the ATP-dependent opening of the channel (14), a process that is mainly controlled by ABP2 (2), the site where the G551D mutation is located. Login to comment 215 ABCC7 p.Gly551Asp All our previous data indicate that ABP2 is not functional in G551D channels. Login to comment 216 ABCC7 p.Gly551Asp We have shown that ATP fails to open the channel and that other nucleotides that act at ABP2 (ADP and AMP-PNP) have no effect on G551D-CFTR (14). Login to comment 217 ABCC7 p.Gly551Asp The reason for this is not known, but because the Gly-551 residue is in the signature motif, which by itself does not bind ATP, nucleotide binding to the head of NBD1 is unlikely to be affected by the G551D mutation. Login to comment 228 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly P-dATP effect on W401G/⌬F508-CFTR and ⌬F508/Y1219G-CFTR. Login to comment 229 ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Gly ABCC7 p.Trp401Phe Representative current traces of W401G/⌬F508 (A) and ⌬F508/Y1219G (B)inthepresenceof50␮M P-dATP.C,summaryofthemaximumcurrent-fold increase in activity induced by 10 ␮M and 50 ␮M P-dATP in W401F/⌬F508 and ⌬F508/Y1219G, and 10 ␮M P-dATP in ⌬F508. Login to comment 233 ABCC7 p.Gly551Asp In this study we show that P-dATP binds to ABP1 to potentiate the activity of G551D-CFTR, and this potentiation is mainly due to a 14-fold increase in the open time of the channels. Login to comment 235 ABCC7 p.Gly551Asp The stabilization of the open channel conformation through nucleotide binding to ABP1 has already been demonstrated for WT (2) and G551D channels (15), but the exact mechanism remains unclear. Login to comment 239 ABCC7 p.Gly551Asp Although for WT-CFTR, ATP hydrolysis may provide the free energy to break NBD dimer apart, it is important to note that, for G551D channels, hydrolysis does not play a role in the closing of the channels (42). Login to comment 241 ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly In support of this notion, the mutation W401G, which likely weakens nucleotide binding to ABP1 (2), decreases the potentiation effect of P-dATP in G551D channels (Fig. 4C). Login to comment 243 ABCC7 p.Gly551Asp However appealing this proposition appears, it is difficult to use the tight-binding argument to explain the prolonged open time of G551D channels in the presence of dATP, because there is no obvious reason for dATP to assume tighter binding in ABP1. Login to comment 244 ABCC7 p.Gly551Asp Indeed, contrary to P-ATP or P-dATP, millimolar dATP is required to potentiate G551D-CFTR. Login to comment 245 ABCC7 p.Gly551Asp Nevertheless, the fact that 3Ј-deoxy-ATP, compared with 2Ј-deoxy-ATP, has a much smaller effect on G551D channels points to a very specific interaction between the ribose of the ATP analog with the amino acids in the binding site. Login to comment 247 ABCC7 p.Gly551Asp As mentioned before, ATP fails to open G551D channels (14). Login to comment 248 ABCC7 p.Gly551Asp The ATP analogs we studied here (dATP and P-dATP) also fail to effectively open G551D channels, because we only observe a moderate increase in the opening rate of the channels. Login to comment 249 ABCC7 p.Tyr1219Gly In fact the mutation Y1219G in ABP2, which greatly decreases the apparent nucleotide-binding affinity at this site, barely diminishes the effect of P-dATP. Login to comment 250 ABCC7 p.Gly551Asp This result indicates that this site is not responsible for most of the potentiation effect by P-dATP in G551D-CFTR. Login to comment 257 ABCC7 p.Gly551Asp Thus, one can envision that, if the opening of G551D-CFTR also involves NBD dimerization, a more hydrophobic ligand in the binding site(s) may facilitate dimerization of the two NBDs. Login to comment 259 ABCC7 p.Gly551Asp In this report we also showed that the potentiation effect of the ATP analogs (P-ATP, dATP, and P-dATP) on G551D-CFTR depends on the interaction of the nucleotide with the head of NBD1 and the signature sequence of NBD2, indicating that the analogs act at the NBD1-NBD2 interface. Login to comment 262 ABCC7 p.Gly551Asp Effect of P-dATP in ⌬F508-CFTR-The dramatic effect of P-dATP on the G551D mutant compelled us to test its effect on other low Po mutants such as ⌬F508-CFTR, the most common CF-causing mutation. Login to comment 269 ABCC7 p.Tyr1219Gly The decrease in the effect of P-dATP in ⌬F508 channels that contain the Y1219G mutation is easier to understand, because this mutation decreases the ATP-binding affinity at ABP2, the site that controls the ATP-dependent opening of the channel, by Ͼ50-fold. Login to comment 270 ABCC7 p.Trp401Gly ABCC7 p.Trp401Gly On the other hand, it was somewhat surprising to observe a reduction of the effect of P-dATP on W401G/⌬F508, because the W401G mutation alone does not affect much the effect of P-dATP on WT-CFTR (see supplemental information). Login to comment 277 ABCC7 p.Gly551Asp The magnitude of the current increase by P-dATP is perhaps the largest increase reported for G551D-CFTR channels so far. Login to comment