ABCMdb

PMID: 25225552

Lin WY, Jih KY, Hwang TC
A single amino acid substitution in CFTR converts ATP to an inhibitory ligand.
J Gen Physiol. 2014 Oct;144(4):311-20. doi: 10.1085/jgp.201411247. Epub 2014 Sep 15., [PubMed]

Sentences

No. Mutations Sentence Comment

10 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp For example, converting the glycine residue at position 551 to an aspartate (i.e., G551D) results in a >100-fold lower open probability (Po) compared with WT-CFTR (Bompadre et al., 2007; Miki et al., 2010). Login to comment 13 ABCC7 p.Gly551Asp As shown to increase the Po of G551D-CFTR by approximately eightfold in vitro (Van Goor et al., 2009), VX-770, when taken orally, significantly improves the symptoms of CF patients carrying such mutation (Accurso et al., 2010; Ramsey et al., 2011). Login to comment 14 ABCC7 p.Gly551Asp Despite these encouraging results, the VX-770-rectified Po of G551D channels is still less A single amino acid substitution in CFTR converts ATP to an inhibitory ligand Wen-Ying Lin,1,2 Kang-Yang Jih,2,3 and Tzyh-Chang Hwang1,2 1 Department of Medical Pharmacology and Physiology, 2 Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211 3 Physician-Scientist Program, National Yang-Ming University, Taipei, 112 Taiwan Cystic fibrosis (CF), one of the most common lethal genetic diseases, is caused by loss-of-function mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel that, when phosphorylated, is gated by ATP. Login to comment 15 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The third most common pathogenic mutation, a glycine-to-aspartate mutation at position 551 or G551D, shows a significantly decreased open probability (Po) caused by failure of the mutant channel to respond to ATP. Recently, a CFTR-targeted drug, VX-770 (Ivacaftor), which potentiates G551D-CFTR function in vitro by boosting its Po, has been approved by the FDA to treat CF patients carrying this mutation. Login to comment 16 ABCC7 p.Gly551Asp Here, we show that, in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit with an unusual time course. Login to comment 17 ABCC7 p.Gly551Asp In marked contrast to wild-type channels, which are stimulated by ATP, sudden removal of ATP in excised inside-out patches elicits an initial increase in macroscopic G551D-CFTR current followed by a slow decrease. Login to comment 18 ABCC7 p.Gly551Asp Furthermore, decreasing [ATP] from 2 mM to 20 &#b5;M resulted in a paradoxical increase in G551D-CFTR current. Login to comment 19 ABCC7 p.Gly551Asp These results suggest that the two ATP-binding sites in the G551D mutant mediate opposite effects on channel gating. Login to comment 20 ABCC7 p.Gly551Asp We introduced mutations that specifically alter ATP-binding affinity in either nucleotide-binding domain (NBD1 or NBD2) into the G551D background and determined that this disease-associated mutation converts site 2, formed by the head subdomain of NBD2 and the tail subdomain of NBD1, into an inhibitory site, whereas site 1 remains stimulatory. Login to comment 21 ABCC7 p.Gly551Ser ABCC7 p.Gly551Glu ABCC7 p.Gly551Lys G551E, but not G551K or G551S, exhibits a similar phenotype, indicating that electrostatic repulsion between the negatively charged side chain of aspartate and the &#e067;-phosphate of ATP accounts for the observed mutational effects. Login to comment 28 ABCC7 p.Gly551Asp Here we showed that in the presence of VX-770, G551D-CFTR becomes responsive to ATP, albeit in a very strange way. Login to comment 29 ABCC7 p.Gly551Asp Once the G551D-CFTR channels in excised inside-out patches are activated by PKA and ATP, upon ATP washout, the macroscopic current shows a biphasic time course: a rapid current increase followed by a slow decay (Fig. 1 C). Login to comment 30 ABCC7 p.Gly551Asp This observation leads us to hypothesize that the G551D mutation converts one of the two ATP-binding sites into an inhibitory site. Login to comment 31 ABCC7 p.Gly551Asp By manipulating ATP-binding affinities at either NBD, we provide evidence that ATP-binding site 2, defined as the one formed by the head subdomain of NBD2 and the tail subdomain of NBD1, assumes an inhibitory function upon ATP binding in G551D-CFTR. Login to comment 32 ABCC7 p.Gly551Ser ABCC7 p.Gly551Glu ABCC7 p.Gly551Lys Because this inhibitory effect is observed also in G551E, but not in G551K or G551S, a basic chemical mechanism of an electrostatic repulsion between the negatively charged side chain of 551D/E and the &#e067;-phosphate of ATP in shaping the observed mutational effects is proposed. Login to comment 36 ABCC7 p.Gly551Asp Therefore, elucidating the molecular mechanism underlying the gating defect of the G551D mutant may lay the foundation for designing second generation, more efficacious CFTR potentiators that may ultimately realize a cure for at least a subset of patients with CF. Login to comment 37 ABCC7 p.Gly551Asp Previous studies have demonstrated that the stimulatory effect of ATP is abolished by the G551D mutation despite a normal surface expression of the mutant proteins (Gregory et al., 1991; Li et al., 1996; Bompadre et al., 2007). Login to comment 38 ABCC7 p.Gly551Asp Although it was speculated that the G-to-D mutation at position 551 likely impedes ATP-induced NBD dimerization because of the critical location of G551 (Lewis et al., 2004; Xu et al., 2014), the underlying mechanism for this gating defect has not been rigorously investigated partly because of the extremely low activity of the G551D channel (Bompadre et al., 2007). Login to comment 50 ABCC7 p.Gly551Asp (C) A real-time current trace of G551D-CFTR channels showing an effect of VX-770 in potentiating the channel activity as well as a biphasic response of the current to ATP washout. Login to comment 52 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly G551D- and G551D/W401G-CFTR and double exponential functions in WT-CFTR using a built-in Levenberg-Marquardt-based algorithm. Login to comment 57 ABCC7 p.Gly551Asp Because of the limited bandwidth of recordings as well as the small signal-to-noise ratio for G551D-CFTR, events shorter than 10 ms (four data points above the half amplitude threshold plus one data point before and one after, for a total of six data points with a 10-ms duration) cannot be measured accurately, resulting in missed events that may contribute partly to the apparent paucity of brief events in the dwell-time histograms shown in Fig. 7 (B and D). Login to comment 58 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly We took a more conservative approach to analyze the single-channel open time for G551D and G551D/Y1219G channels as only current traces with up to two opening steps were used. Login to comment 65 ABCC7 p.Gly551Asp Thus, it seems unsurprising that previous studies show that ATP fails to increase the activity of G551D-CFTR in excised inside-out patches (Bompadre et al., 2007; Miki et al., 2010). Login to comment 66 ABCC7 p.Gly551Asp Unexpectedly however, in the presence of VX-770, removal of ATP resulted in a sudden increase of the G551D-CFTR current followed by a slow decay (Figs. 1 C and 2 A). Login to comment 83 ABCC7 p.Gly551Asp To obtain macroscopic currents of G551D-CFTR for our kinetic studies, we applied 200 nM VX-770 in all experiments except the one shown in Fig. 3 C. Login to comment 97 ABCC7 p.Gly551Asp We therefore chose to test two more [ATP] values on G551D-CFTR in addition to 2 mM that is likely to saturate both ATP-binding sites: 20 &#b5;M, which presumably allows a full occupancy at site 1 but suboptimal binding of ATP to site 2, and 1 &#b5;M, which may ensure minimal binding of ATP to both sites. Login to comment 98 ABCC7 p.Gly551Asp Results shown in Fig. 3 (A and B) indeed confirm our prediction: the G551D-CFTR current is increased when [ATP] is decreased from 2 mM to 20 &#b5;M (Fig. 3 A). Login to comment 100 ABCC7 p.Gly551Asp As all the results shown so far were conducted in the presence of VX-770, we have to ask whether this inhibitory action of ATP is also seen with G551D-CFTR in the absence of VX-770. Login to comment 101 ABCC7 p.Gly551Asp It is technically challenging to conduct similar experiments in the absence of VX-770 because the open probability of G551D-CFTR is too low (Bompadre et al., 2007; Miki et al., 2010) to acquire patches yielding macroscopic currents. Login to comment 102 ABCC7 p.Gly551Asp Once we are limited to small G551D-CFTR currents (<5 pA) in the absence of VX-770, a large current fluctuation caused by a time constant of 29.6 &#b1; 1.8 s (n = 8). Login to comment 103 ABCC7 p.Gly551Asp Interestingly, this long time constant is indistinguishable from the time constant of the current decay seen with the G551D channels (31.1 &#b1; 5.3 s; n = 12; Fig. 2 A, red line). Login to comment 106 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The major difference between WTand G551D-CFTR is that one normally stimulatory action of ATP is reversed in G551D channels, whereas the other one remains relatively unchanged. Login to comment 107 ABCC7 p.Gly551Asp The biphasic response upon ATP removal presented in Fig. 2 can thus be explained by a simple idea that the G551D mutation converts one of the stimulatory sites of ATP to an inhibitory site. Login to comment 109 ABCC7 p.Gly551Asp First, a decrease of [ATP] may first increase the G551D-CFTR current as the result of a lowered probability of occupancy for this presumed inhibitory site, but further lowering [ATP] will dampen the current as ATP binding to the stimulatory site is jeopardized. Login to comment 110 ABCC7 p.Gly551Asp Second, mutations that alter ATP-binding affini- tiesattheresponsibleATP-bindingsitemaydifferentially perturb the inhibitory or stimulatory action of ATP on G551D-CFTR. Login to comment 111 ABCC7 p.Gly551Asp A simple way to test the first prediction is to measure G551D-CFTR currents at different [ATP]. Login to comment 114 ABCC7 p.Gly551Asp This result Figure 2.ߓ The G551D mutation alters the time course of current decay upon washout of ATP. Login to comment 115 ABCC7 p.Gly551Asp (A and B) Biphasic changes of macroscopic currents upon ATP removal in the presence of VX-770 for G551D-CFTR (A) and WT-CFTR (B). Login to comment 116 ABCC7 p.Gly551Asp (A) In the continuous presence of VX-770, addition of PKA and ATP slowly activated macroscopic G551D-CFTR currents to a steady-state. Login to comment 124 ABCC7 p.Gly551Asp ATP (Zhou et al., 2006), were introduced into the G551D background. Login to comment 126 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly Also consistent with our hypothesis, removing the entire side chain, i.e., the G551D/Y1219G mutation, completely obliterates the rapid current increasing phase (Fig. 4 C) upon ATP removal as if only minimal occupancy of site 2 takes place at 2 mM [ATP]. Login to comment 127 ABCC7 p.Gly551Asp These graded changes in the inhibitory action of ATP on G551D mutants echo the reported graded changes in ATP-binding affinity for the stimulatory action of ATP on WT-CFTR (Zhou et al., 2006). Login to comment 128 ABCC7 p.Gly551Asp Thus, site 2, the main gating site for WT-CFTR, becomes inhibitory in G551D-CFTR! Login to comment 130 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Nonetheless, we were able to reproduce the reverse [ATP] dependence of G551D-CFTR currents in the absence of VX-770 (Fig. 3 C), suggesting that the inhibitory action of ATP on site 2 is an intrinsic property of the G551D mutation. Login to comment 132 ABCC7 p.Gly551Asp To further test the hypothesis that in G551D-CFTR site 2 becomes inhibitory, we manipulated ATP-binding affinity at site 2 by altering the aromatic amino acid Y1219 that has been shown both structurally and functionally (Zhou et al., 2006; PDG ID of NBD2: 3GD7) to play a pivotal role in ATP binding in the head subdomain of NBD2. Login to comment 133 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe Y1219F, Y1219I, and Y1219G, mutations known to cause a graded change of the apparent affinity for Figure 3.ߓ Paradoxical [ATP] dependence of G551D-CFTR currents supports the hypothesis of two ATP-binding sites exerting opposite actions. Login to comment 134 ABCC7 p.Gly551Asp (A) Reversed ATP-dose dependence in G551D-CFTR. Login to comment 135 ABCC7 p.Gly551Asp A switch from 2 mM to 20 &#b5;M ATP increased the current of G551D-CFTR, and the effect readily reversed once the ATP concentration was increased. Login to comment 141 ABCC7 p.Gly551Asp (B) A continuous recording of G551D-CFTR showing bell-shaped [ATP] dependence. Login to comment 143 ABCC7 p.Gly551Asp (C) Inverse [ATP] dependence of G551D-CFTR currents in the absence of VX-770. Login to comment 146 ABCC7 p.Gly551Asp (D) Fold increase of G551D-CFTR currents upon switching solution from 2 mM to 20 &#b5;M ATP (I20&#b5;M/I2mM) in the presence or absence of VX-770. Login to comment 148 ABCC7 p.Gly551Asp Interestingly, we noticed that the raw current traces in Van Goor et al. (2009) indeed suggest the existence of at least two different opening events for G551D-CFTR in the presence of VX-770. Login to comment 149 ABCC7 p.Gly551Asp To further test this idea, we recorded microscopic currents of G551D-CFTR in the presence of 20 &#b5;M ATP (Fig. 7 A), a concentration meant to keep a minimum occupancy of site 2 but at the same time an occupied site 1. Login to comment 151 ABCC7 p.Gly551Asp Fig. 7 A shows samples of raw current traces of G551D-CFTR at 20 &#b5;M ATP. Login to comment 154 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly Similar observations were made for G551D/Y1219G-CFTR, in which 20 &#b5;M ATP is expected to bear negligible ATP occupancy at site 2 (Fig. 7, C and D). Login to comment 155 ABCC7 p.Gly551Asp Thus, even for monoliganded G551D channels, there exist two distinct open states, an observation which is inconsistent with the modified model depicted in Fig. 6 D. Login to comment 159 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly Of note, the time constant of slow current decay phase in G551D/ Y1219G-CFTR was unchanged compared with G551D-CFTR (Figs. 4 C and 5 B), indicating that this slow current decay is not controlled by ATP dissociation from site 2. Login to comment 162 ABCC7 p.Gly551Ser ABCC7 p.Gly551Lys With a neutral (G551S) or cationic (G551K) side chain at residue 551, the channels respond to ATP in a manner similar to that of WT-CFTR. Login to comment 164 ABCC7 p.Gly551Asp ABCC7 p.Gly551Glu In contrast, G551E-CFTR channels behave just like G551D-CFTR (Fig. 6 C), suggesting that an anionic side chain at residue 551 is required to confer this inhibitory action to site 2. Login to comment 165 ABCC7 p.Gly551Asp Collectively, we modified the gating scheme originally proposed for WT-CFTR (Jih et al., 2012) by eliminating NBD-dimerized states (Fig. 6 D) to explain G551D-induced gating defects. Login to comment 166 ABCC7 p.Gly551Asp But, is the remaining four-state scheme (Fig. 6 D) sufficient to explain G551D-CFTR gating? Login to comment 167 ABCC7 p.Gly551Asp Note there are two open states ("monoliganded" and "biliganded") in Figure 4.ߓ Effects of mutations at Y1219 on the response of G551D-CFTR to ATP washout. Login to comment 168 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly ABCC7 p.Tyr1219Ile ABCC7 p.Tyr1219Phe (A-C) Real-time current traces in response to ATP removal for G551D/Y1219F (A), G551D/Y1219I (B), and G551D/Y1219G (C). Login to comment 169 ABCC7 p.Gly551Asp (D) Summary of the ratios between the peak current after ATP washout (IPEAK) and the steady-state current before ATP removal (IATP) for G551D and its variants. Login to comment 170 ABCC7 p.Gly551Asp Mean &#b1; SEM is shown. this chemical mechanism that is also supported by recent computational work (Xu et al., 2014), it seems safe to discount any role of NBD dimerization in G551D-CFTR gating. Login to comment 171 ABCC7 p.Gly551Asp We therefore remove the NBD-dimerized states in Fig. 6 D for the G551D mutant (compare Bompadre et al., 2007). Login to comment 173 ABCC7 p.Gly551Asp However, the remaining four-state scheme (Fig. 6 D) underpinning the gating mechanism for G551D-CFTR may still not suffice for the following reasons. Login to comment 174 ABCC7 p.Gly551Asp First, it obviously fails to explain the slow decay phase seen in both WTand G551D-CFTR (Fig. 2). Login to comment 181 ABCC7 p.Gly551Asp The data presented in the current manuscript support the notion that this functionally important site 2 is converted to an inhibitory site in G551D-CFTR, a conclusion independent of any kinetic model for CFTR gating. Login to comment 183 ABCC7 p.Gly551Asp Once we accept Figure 5.ߓ Characterization of the slow current decay in G551D-CFTR. Login to comment 184 ABCC7 p.Trp401Gly (A) Acceleration of the slow-phase current decay by the W401G mutation. Login to comment 185 ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly A continuous recording demonstrates an unequivocal biphasic response upon ATP washout for G551D/ W401G-CFTR. Login to comment 186 ABCC7 p.Gly551Asp The current decay phase was fitted with a single exponential function (red line), yielding a time constant of 17.6 s, which is significantly shorter than that of G551D-CFTR (P < 0.05). Login to comment 188 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Trp401Gly ABCC7 p.Tyr1219Gly 31.1 &#b1; 5.3 s (n = 12) for G551D-CFTR, 19.0 &#b1; 3.2 s (n = 8) for G551D/W401G-CFTR, and 31.7 &#b1; 5.9 s (n = 12) for G551D/Y1219G-CFTR. Login to comment 191 ABCC7 p.Gly551Ser ABCC7 p.Gly551Glu ABCC7 p.Gly551Lys (A-C) Responses to ATP withdrawal in different G551 mutants: G551S (A), G551K (B), and G551E (C). Login to comment 192 ABCC7 p.Gly551Asp (D) A modified gating model with NBD-dimerized states crossed out for G551D-CFTR. Login to comment 194 ABCC7 p.Gly551Asp "D" in the red star depicts the location of G551D in the tail subdomain of NBD1. Login to comment 201 ABCC7 p.Gly551Asp Once the O0 state is added to the scheme, the inhibitory action of ATP in G551D-CFTR can be explained by a trapping of the channel in the C2 ATP state in the presence of millimolar ATP; the removal of ATP allows the channel to travel to the more stable O0 state, causing an increase of the current. Login to comment 204 ABCC7 p.Gly551Asp As described in the Results, this inhibitory action of ATP on G551D-CFTR is also present in the absence of VX-770 (Fig. 3 C). Login to comment 205 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The microscopic nature of G551D-CFTR conditions are designed to assess the gating behavior of monoliganded G551D-CFTR (Fig. 7). Login to comment 206 ABCC7 p.Gly551Asp But, once this simple equilibrium gating model is rejected, how can we explain kinetically the inhibitory effect of ATP binding on site 2 of G551D-CFTR channels? Login to comment 208 ABCC7 p.Gly551Asp This energetic coupling between NBD dimerization and gate opening predicts that the O2 state in the G551D-CFTR should also favor dimerization of its NBDs, which, if it occurs, likely will proceed very slowly without a bound ATP molecule in site 2. Login to comment 209 ABCC7 p.Gly551Asp By simply adding another open state (O0) depicting an open channel conformation with a dimerized NBDs but an empty site 2, we can provide a tentative explanation for not only microscopic data shown in Fig. 7, but also ATP-dependent inhibition of macroscopic G551D-CFTR currents. Login to comment 211 ABCC7 p.Gly551Asp (A) Single-channel recording of G551D-CFTR in the presence of 20 &#b5;M ATP. Login to comment 212 ABCC7 p.Gly551Asp (B) Open time histogram for G551D-CFTR. Login to comment 214 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly (C) Single-channel recording of G551D/Y1219G-CFTR in the presence of 20 &#b5;M ATP. Login to comment 215 ABCC7 p.Gly551Asp ABCC7 p.Tyr1219Gly (D) Open time histogram for G551D/Y1219G-CFTR. Login to comment 228 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. Login to comment 230 ABCC7 p.Gly551Asp ABCC7 p.Arg117His A little CFTR goes a long way: CFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor. Login to comment 260 ABCC7 p.Gly551Asp However, it puzzles us as to why the inverse [ATP] dependence of G551D-CFTR (Fig. 3 C) was never reported in the past, although a recent study (Xu et al., 2014) did characterize this mutant CFTR at different [ATP]. Login to comment 261 ABCC7 p.Gly551Asp We noted that in Xu et al. (2014), 0.1, 1, and 5 mM [ATP] were tested on G551D-CFTR in the absence of VX-770. Login to comment 263 ABCC7 p.Gly551Asp In addition to offering some more insights into the gating mechanism of CFTR, our work could also shed light on possible new strategies for developing novel therapies for patients carrying the G551D mutation. Login to comment 264 ABCC7 p.Gly551Asp Although VX-770 has been approved by the FDA for the treatment of CF patients carrying the G551D mutation, it may be still a long way to a complete cure. Login to comment 265 ABCC7 p.Gly551Asp Using cAMP-dependent sweating as an in vivo measurement for CFTR activity, Char et al. (2014) estimated VX-770-rectified G551D-CFTR function to be &#e07a;5% of the WT mean. Login to comment 266 ABCC7 p.Gly551Asp Thus, a further improvement of G551D-CFTR activity with compounds that can complement the action of VX-770 is expected to benefit these CF patients currently taking Ivacaftor. Login to comment 267 ABCC7 p.Gly551Asp Our demonstration of a significant inhibition of G551D-CFTR by ATP opens the door for a new strategy of drug development. Login to comment 268 ABCC7 p.Gly551Asp By designing reagents that can compete off ATP binding to site 2, we expect a doubling of G551D-CFTR function by eliminating this inhibitory action of ATP. Login to comment 278 ABCC7 p.Gly551Asp Effect of VX-770 in persons with cystic fibrosis and the G551D-CFTR mutation. N. Engl. J. Med. 363:1991-2003. http:// dx.doi.org/10.1056/NEJMoa0909825 Smith, P.C., N. Karpowich, L. Millen, J.E. Moody, J. Rosen, P.J. Thomas, and J.F. Hunt. Login to comment 305 ABCC7 p.Gly551Asp Revertant mutants modify, but do not rescue, the gating defect of the cystic fibrosis mutant G551D-CFTR. Login to comment 341 ABCC7 p.Gly551Asp A CFTR potentiator in patients with cystic fibrosis and the G551D mutation. N. Engl. J. Med. 365:1663-1672. http://dx.doi.org/10.1056/NEJMoa1105185 Ren, X.-Q., T. Furukawa, M. Haraguchi, T. Sumizawa, S. Aoki, M. Kobayashi, and S. Akiyama. Login to comment