ABCMdb

PMID: 25148434

Liu X, Dawson DC
Cystic fibrosis transmembrane conductance regulator (CFTR) potentiators protect G551D but not DeltaF508 CFTR from thermal instability.
Biochemistry. 2014 Sep 9;53(35):5613-8. doi: 10.1021/bi501007v. Epub 2014 Aug 22., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiators Protect G551D but Not ƊF508 CFTR from Thermal Instability Xuehong Liu*,ߤ and David C. Dawson Department of Physiology & Pharmacology, Oregon Health & Science University, Portland, Oregon 97239, United States ABSTRACT: The G551D cystic fibrosis transmembrane conductance regulator (CFTR) mutation is associated with severe disease in ~5% of cystic fibrosis patients worldwide. Login to comment 3 ABCC7 p.Gly551Asp Recent clinical trials demonstrated a beneficial effect of the CFTR potentiator, Ivacaftor (VX-770), on lung function of patients bearing at least one copy of G551D CFTR, but no comparable effect on ƊF508 homozygotes. Login to comment 6 ABCC7 p.Gly551Asp This additional mutant phenotype raised the possibility that the differences in the behavior of ƊF508 and G551D CFTR, as well as the disparate efficacy of Ivacaftor, might be a reflection of the differing thermal stabilities of the two channels at 37 &#b0;C. Login to comment 7 ABCC7 p.Gly551Asp We compared the thermal stability of G551D and ƊF508 CFTR in Xenopus oocytes in the presence and absence of CTFR potentiators. Login to comment 8 ABCC7 p.Gly551Asp G551D CFTR exhibited a thermal instability that was comparable to that of ƊF508 CFTR. Login to comment 9 ABCC7 p.Gly551Asp G551D CFTR, however, was protected from thermal instability by CFTR potentiators, whereas ƊF508 CFTR was not. Login to comment 10 ABCC7 p.Gly551Asp These results suggest that the efficacy of VX-770 in patients bearing the G551D mutation is due, at least in part, to the ability of the small molecule to protect the mutant channel from thermal instability at human body temperature. Login to comment 11 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The recent demonstration of efficacy of a CFTR potentiator in patients carrying at least one copy of G551D CFTR was a quantum leap for CF therapy, being the first instance of a therapeutic intervention based on a small molecule that directly targets the mutant gene product.1-4 The potentiator, VX-770, known as Ivacaftor or Kalydeco, did not exhibit similar efficacy in patients homozygous for the more common mutation, ƊF508, however.5 This difference could be attributed to the well-established difference in the molecular phenotypes of the two mutations, namely, a gating defect for G551D and a combined trafficking and gating defect for ƊF508,6 but we wondered if the two mutants might also differ with regard to the more recently established mutant CFTR phenotype of thermal instability. Login to comment 15 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The apparent disparity in clinical efficacy of VX-770 in compound heterozygotes (ƊF508/G551D) carrying one copy of G551D CFTR2,4 and a G551D homozygote carrying two copies of G551D CFTR,3 as well as the modest efficacy of VX-770 seen in ƊF508 homozygotes, suggested to us that ƊF508 CFTR channels and G551D CFTR channels might differ in their thermal stabilities. Login to comment 16 ABCC7 p.Gly551Asp Might it be, for example, that the well-known trafficking defect seen with ƊF508 CFTR is, at least in part, a reflection of thermal instability apparent in a channel function assay, a thermal instability that might be lacking in the normally trafficked G551D channels? Login to comment 17 ABCC7 p.Gly551Asp We compared the thermal stability of G551D CFTR channels expressed in Xenopus oocytes with that previously reported by us for ƊF508 CFTR channels. Login to comment 18 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp We found, contrary to our initial expectations, that G551D CFTR channel function was thermally unstable at 37 &#b0;C, although G551D CFTR channel behavior differed from that of the ƊF508 channels in several important respects. Login to comment 20 ABCC7 p.Gly551Asp Second, following a 37 &#b0;C thermal challenge, the conductance due to G551D channels recovered almost fully (85%), in contrast to that seen with ƊF508 channels, which Received: August 12, 2014 Published: August 13, 2014 Article pubs.acs.org/biochemistry (c) 2014 American Chemical Society 5613 dx.doi.org/10.1021/bi501007v | Biochemistry 2014, 53, 5613-5618 This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Login to comment 22 ABCC7 p.Gly551Asp Most importantly, however, G551D CFTR channels appeared to be protected from thermal instability at 37 &#b0;C by CFTR potentiators, including VX-770. Login to comment 23 ABCC7 p.Gly551Asp Furthermore, potentiators also provoked an increase in conductance due to G551D channels at 37 &#b0;C, following thermal deactivation, a condition more like that in vivo. Login to comment 24 ABCC7 p.Gly551Asp These results provide a mechanistic basis for the differing efficacy of VX-770 in patients carrying G551D and ƊF508 CFTR and have important implications for the design of assays for small molecule screening. Login to comment 45 ABCC7 p.Gly551Asp a0; RESULTS G551D Channels Exhibit Thermal Instability. Login to comment 46 ABCC7 p.Gly551Asp Figure 1A shows the results of a representative experiment in which an oocyte expressing G551D CFTR channels was exposed to an increase in temperature from 22 to 37 &#b0;C for 10 min. Login to comment 48 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp However, there was a distinct, quantitative difference between the thermal deactivation of G551D CFTR and that we previously reported for ƊF508 CFTR.7 The rate of thermal deactivation was more rapid for the G551D channels [t1/2 = 0.3 min (&#b1;0.04; n = 3) vs 3.7 min (&#b1;0.3; n = 11) for ƊF508 channels]7 (P < 0.05). Login to comment 49 ABCC7 p.Gly551Asp More striking, however, was the greater extent of recovery following the thermal challenge, 85 &#b1; 15% (n = 3) for G551D and 43 &#b1; 15% (n = 3) for ƊF5087 (P < 0.05). Login to comment 50 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Ile539Thr ABCC7 p.Ile539Thr The relatively rapid and nearly complete recovery of the G551D channels was reminiscent of that seen with ƊF508 channels when the phenylalanine deletion was combined with a nearby, second-site suppressor mutation like I539T (ƊF508/I539T CFTR).7 Upon comparison to contemporaneous experiments with ƊF508 CFTR channels (see Figures 3 and 4), it also appeared that the extent of thermal deactivation was lower for G551D channels. Login to comment 52 ABCC7 p.Gly551Asp Thermal instability of G551D CFTR channels. Login to comment 53 ABCC7 p.Gly551Asp (A) Following stimulation [10 bc;M isoproterenol and 1 mM IBMX (hatched bar and crosshairs)], an oocyte expressing G551D CFTR was warmed to 37 &#b0;C (gray bar and circles) for 10 min. Login to comment 55 ABCC7 p.Gly551Asp (B) Summary of G551D CFTR conductance before and after thermal deactivation (P < 0.05). Login to comment 59 ABCC7 p.Gly551Asp The half-time for G551D CFTR activation averaged 37.2 &#b1; 4 min (n = 18), and the half-time for ƊF508 CFTR activation under comparable conditions averaged 7.0 &#b1; 2.0 min (n = 18). Login to comment 60 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp A prolonged half-time for activation by cAMP is a consistent feature of G551D channels and is compatible with the long closed times seen in single-channel recordings.25 conductance at 22 &#b0;C, averaged 40 &#b1; 8.1% (n = 7) for G551D and 7 &#b1; 1.4% (n = 6) for ƊF508. Login to comment 61 ABCC7 p.Gly551Asp Taken together, these results suggested that G551D CFTR channels, despite their rapid, thermal deactivation, are less severely impacted by the elevated temperature than ƊF508 channels. Login to comment 62 ABCC7 p.Gly551Asp CFTR Potentiators Protect G551D CFTR Channels but Not ƊF508 CFTR Channels from Thermal Deactivation. Login to comment 63 ABCC7 p.Gly551Asp The unexpected finding of thermal instability of G551D channel function prompted us to compare the effects of CFTR potentiators on the two constructs during a 37 &#b0;C thermal challenge. Login to comment 67 ABCC7 p.Gly551Asp Differential effects of CFTR potentiators on the thermal instability of G551D CFTR channels. Login to comment 68 ABCC7 p.Gly551Asp (A) Following stimulation at 22 &#b0;C, an oocyte expressing G551D CFTR was warmed to 37 &#b0;C (gray bar and circles) and then exposed to 10 bc;M CF172 at 37 &#b0;C. Login to comment 70 ABCC7 p.Gly551Asp (B-E) Following stimulation, oocytes expressing G551D CFTR were exposed to 10 bc;M VX-770, 10 bc;M P2 (PG-01), 50 bc;M Genistein, and 10 bc;M P1, respectively, warmed to 37 &#b0;C (gray bar and circles) in the presence of potentiators, and then exposed to 10 bc;M CF172 at 37 &#b0;C. Login to comment 72 ABCC7 p.Gly551Asp Doses of the potentiators were chosen on the basis of the maximal concentration reported in the literature as well as our previous study.7,16,26-28 With respect to the activated conductance prior to the application of potentiator, the P values for stimulation by Genistein, P1, P2, and VX770 are all <0.05. similar experiments employing oocytes expressing G551D channels produced dramatically different results. Login to comment 73 ABCC7 p.Gly551Asp Panels A-E of Figure 2 compare representative experiments in which we tested the effects of four CFTR potentiators, VX-770, P1, P2, and Genistein, on the thermal stability of conductance due to G551D CFTR channels. Login to comment 74 ABCC7 p.Gly551Asp As summarized in Figure 2F via comparison of the conductance near the termination of the thermal pulse with that seen prior to exposure to a potentiator, all four potentiators protected G551D CFTR channels from thermal deactivation, albeit to varying extents. Login to comment 76 ABCC7 p.Gly551Asp Also, note that the steady state-activated conductance due to G551D CFTR channels exhibited substantial variation from oocyte to oocyte. Login to comment 83 ABCC7 p.Gly551Asp The results described above point to an important difference in the effect of CFTR potentiators like VX-770 on G551D and ƊF508 CFTR channels that may have a counterpart in the efficacy of VX-770 in patients carrying these mutations. Login to comment 84 ABCC7 p.Gly551Asp In a patient`s first encounter with the drug, however, any G551D CFTR channels would presumably be partially deactivated, raising the question of the efficacy of potentiators in channels previously inactivated by exposure to 37 &#b0;C. Login to comment 85 ABCC7 p.Gly551Asp Panels A and B of Figure 4 show the results of representative experiments in which we investigated the efficacy of the most potent potentiator, VX-770, in oocytes expressing G551D CFTR channels when the drug was applied following the completion of thermal deactivation at 37 &#b0;C, to better mimic the in vivo condition. Login to comment 86 ABCC7 p.Gly551Asp Exposure to VX-770 induced a robust increase in the conductance due to G551D channels at 37 &#b0;C (Figure 4A,C). Login to comment 88 ABCC7 p.Gly551Asp Comparison of the data summarized in Figure 4 with that in Figure 2 suggests that VX-770, when applied at 37 &#b0;C, not only "protected" the G551D CFTR channels but also increased conductance beyond that seen at 22 &#b0;C. Login to comment 90 ABCC7 p.Gly551Asp Figure 4D summarizes the efficacy of VX-770 in G551D and ƊF508 CFR channels at 37 &#b0;C. Login to comment 100 ABCC7 p.Gly551Asp VX-770 stimulated G551D but not ƊF508 CFTR channels at 37 &#b0;C. Login to comment 101 ABCC7 p.Gly551Asp (A) Following activation of conductance via an increase in intracellular cAMP concentration (see Materials and Methods), an oocyte expressing G551D CFTR was warmed to 37 &#b0;C (gray bar and circles). Login to comment 104 ABCC7 p.Gly551Asp (C) Summary of the conductance due to G551D CFTR at 37 &#b0;C before and after stimulation by 10 bc;M VX770. Login to comment 109 ABCC7 p.Gly551Asp G551D channels, like ƊF508 channels, exhibit thermal instability. Login to comment 110 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp It would seem appropriate, therefore, to add assays of the thermal stability of channel function to those currently used to screen chemical libraries for efficacious compounds; that is, screening should include assays for "protection" from thermal instability, and compound efficacy should be compared at 37 &#b0;C. Yu et al.6 reported that, in membrane patches detached from FRT cells at room temperature, Ivacaftor (VX-770) was effective in increasing the open probability of a variety of CFTR channel constructs bearing mutations that adversely affected channel gating, consistent with a previous report of an increase in the open probability of ƊF508 CFTR channels under similar conditions.16 However, in FRT cell layers assayed in Ussing chambers at 37 &#b0;C, the efficacy of Ivacaftor on conductance due to ƊF508 CFTR channels was minimal, whereas a robust activation of conductance due to G551D channels was detected, despite a minimal conductance prior to exposure to Ivacaftor.6 It seems clear that CFTR potentiators, compounds selected for their ability to increase CFTR channel open probability, can, among other things, stabilize the activated state of G551D CFTR channels at 37 &#b0;C, an effect that may provide clues about the mode of action of these compounds and ultimately their binding sites. Login to comment 114 ABCC7 p.Gly551Asp Clearly, G551D CFTR channels, which are trafficked normally or nearly normally, nevertheless exhibit severe thermal instability of channel function. Login to comment 115 ABCC7 p.Gly551Asp On the other hand, the difference between G551D and ƊF508 CFTR could simply be one of degree. Login to comment 116 ABCC7 p.Gly551Asp It may be that the G551D CFTR channels, although thermally unstable, may not proceed as far along the unfolding pathway at 37 &#b0;C as do the ƊF508 channels. Login to comment 117 ABCC7 p.Gly551Asp G551D CFTR channels would, thereby, escape intracellular degradation but nevertheless fail to contribute adequately to chloride conductance due to the thermal instability of channel function that seems likely to reduce channel open probability at 37 &#b0;C. Login to comment