ABCMdb

PMID: 22293084

Yu H, Burton B, Huang CJ, Worley J, Cao D, Johnson JP Jr, Urrutia A, Joubran J, Seepersaud S, Sussky K, Hoffman BJ, Van Goor F
Ivacaftor potentiation of multiple CFTR channels with gating mutations.
J Cyst Fibros. 2012 May;11(3):237-45. doi: 10.1016/j.jcf.2011.12.005. Epub 2012 Jan 30., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Original Article Ivacaftor potentiation of multiple CFTR channels with gating mutations Haihui Yu, Bill Burton, Chien-Jung Huang, Jennings Worley, Dong Cao, James P. Johnson Jr., Art Urrutia, John Joubran, Sheila Seepersaud, Katherine Sussky, Beth J. Hoffman, Fredrick Van Goor ⁎ Vertex Pharmaceuticals Incorporated, 130 Waverly St, Cambridge, MA 02139, United States Received 20 October 2011; received in revised form 21 December 2011; accepted 27 December 2011 Available online 30 January 2012 Abstract Background: The investigational CFTR potentiator ivacaftor (VX-770) increased CFTR channel activity and improved lung function in subjects with CF who have the G551D CFTR gating mutation. Login to comment 4 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg These included the G551D, G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P and G1349D CFTR gating mutations. Login to comment 5 ABCC7 p.Gly551Asp Conclusion: These in vitro data suggest that ivacaftor has a similar effect on all CFTR forms with gating defects and support investigation of the potential clinical benefit of ivacaftor in CF patients who have CFTR gating mutations beyond G551D. Login to comment 22 ABCC7 p.Gly551Asp The most common CFTR gating mutation in patients with CF is G551D [6-7]. Login to comment 23 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Other known CFTR gating mutations include G178R, G551S, G970R, G1244E, S1255P, and G1349D [9-11]. Login to comment 24 ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Other known CFTR gating mutations include G178R, G551S, G970R, G1244E, S1255P, and G1349D [9-11]. Login to comment 28 ABCC7 p.Gly551Asp Ivacaftor (VX-770), an investigational CFTR potentiator [12], increased the channel open probability of G551D-CFTR to enhance chloride transport in vitro [12]. Login to comment 29 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp In patients with CF who have the G551D mutation, ivacaftor increased clinical measures of chloride transport through CFTR, and improved clinical measures of lung function [13]. Login to comment 30 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Ivacaftor has also been shown to potentiate normal CFTR and F508del-CFTR, suggesting that the compound is not specific for G551D-CFTR. Login to comment 31 ABCC7 p.Gly551Asp Ivacaftor has also been shown to potentiate normal CFTR and F508del-CFTR, suggesting that the compound is not specific for G551D-CFTR. Login to comment 34 ABCC7 p.Gly551Asp These in vitro data support the investigation of the clinical benefit of CFTR potentiators such as ivacaftor, in patients with CF who have CFTR gating mutations beyond G551D. Login to comment 35 ABCC7 p.Gly551Asp These in vitro data support the investigation of the clinical benefit of CFTR potentiators such as ivacaftor, in patients with CF who have CFTR gating mutations beyond G551D. Login to comment 39 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg These included G551D-, G178R-, S549N-, S549R-, G551S-, G970R-, G1244E-, S1251N-, S1255P-, and G1349D-CFTR [4,7,9-11]. Login to comment 40 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg These included G551D-, G178R-, S549N-, S549R-, G551S-, G970R-, G1244E-, S1251N-, S1255P-, and G1349D-CFTR [4,7,9-11]. Login to comment 46 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg This analysis showed that, as expected for known CFTR gating mutations (G551D, G178R, G551S, G970R, G1244E, S1255P, and G1349D) [5,9-11], the amount of CFTR delivered to the cell surface was generally similar between CFTR with gating defects and normal CFTR. Login to comment 47 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg This analysis showed that, as expected for known CFTR gating mutations (G551D, G178R, G551S, G970R, G1244E, S1255P, and G1349D) [5,9-11], the amount of CFTR delivered to the cell surface was generally similar between CFTR with gating defects and normal CFTR. Login to comment 48 ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser Interestingly, there was significantly more G551S-CFTR at the cell surface than normal CFTR, although the total amount of G551S-CFTR synthesized and the ratio of mature to total CFTR were similar to normal CFTR. Login to comment 49 ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser Interestingly, there was significantly more G551S-CFTR at the cell surface than normal CFTR, although the total amount of G551S-CFTR synthesized and the ratio of mature to total CFTR were similar to normal CFTR. Login to comment 50 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Ivacaftor increased the channel gating of mutant CFTR with defective channel gating The effect of ivacaftor on CFTR channel gating was monitored by quantifying the channel open probability by patch-clamp electrophysiology using membrane patches excised from FRT cells expressing the known CFTR gating mutations, G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, or G1349D-CFTR. Login to comment 51 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Ivacaftor increased the channel gating of mutant CFTR with defective channel gating The effect of ivacaftor on CFTR channel gating was monitored by quantifying the channel open probability by patch-clamp electrophysiology using membrane patches excised from FRT cells expressing the known CFTR gating mutations, G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, or G1349D-CFTR. Login to comment 52 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Under these conditions, the baseline CFTR channel open probability of G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR was ≤5% of normal CFTR (Fig. 2, B; Table 1). Login to comment 53 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg For most mutant CFTR forms, the single channel current amplitude, a measure of channel conductance, was similar to normal CFTR (between 77% and 122% of normal CFTR), although a small but statistically significant difference in single channel current amplitude was observed for S1255P-CFTR (Table 1). Login to comment 54 ABCC7 p.Ser1255Pro For most mutant CFTR forms, the single channel current amplitude, a measure of channel conductance, was similar to normal CFTR (between 77% and 122% of normal CFTR), although a small but statistically significant difference in single channel current amplitude was observed for S1255P-CFTR (Table 1). Login to comment 56 ABCC7 p.Gly551Asp These data indicated that ivacaftor increased the channel open probability of all mutant CFTR forms with defective channel gating tested to a similar or greater extent than G551D-CFTR. Login to comment 57 ABCC7 p.Gly551Asp These data indicated that ivacaftor increased the channel open probability of all mutant CFTR forms with defective channel gating tested to a similar or greater extent than G551D-CFTR. Login to comment 58 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Ivacaftor enhanced chloride transport through mutant CFTR with defective channel gating The impact of the increase in CFTR channel gating by ivacaftor on total chloride transport was assessed in Ussing chamber studies using FRT cells expressing the known CFTR gating mutations, G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR. Login to comment 59 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Ivacaftor enhanced chloride transport through mutant CFTR with defective channel gating The impact of the increase in CFTR channel gating by ivacaftor on total chloride transport was assessed in Ussing chamber studies using FRT cells expressing the known CFTR gating mutations, G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR. Login to comment 61 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly178Arg Under these conditions, the baseline level of chloride transport in FRT cells expressing G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR was b10% of normal CFTR (Fig. 3; Table 2), which was consistent with the low CFTR channel open probability of these mutant CFTR forms (Table 1). Login to comment 62 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly178Arg Under these conditions, the baseline level of chloride transport in FRT cells expressing G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR was b10% of normal CFTR (Fig. 3; Table 2), which was consistent with the low CFTR channel open probability of these mutant CFTR forms (Table 1). Login to comment 64 ABCC7 p.Gly551Asp The EC50 of ivacaftor for all mutant CFTR forms tested was similar to G551D-CFTR (range: 161 to 594 nM) (Fig. 3C; Table 2). Login to comment 65 ABCC7 p.Gly551Asp The EC50 of ivacaftor for all mutant CFTR forms tested was similar to G551D-CFTR (range: 161 to 594 nM) (Fig. 3C; Table 2). Login to comment 67 ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn The CF-associated CFTR mutations, S549N, S549R, and S1251N, are located in the ABC signature sequence (Fig. 4; LSGGQ; S549N,R in NBD1) and Walker A motif (Fig. 4; S1251N in NBD2), that form the ATP binding sites [14]. Login to comment 68 ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn Immunoblot and Ussing chamber studies indicated that, although the maturation of S549N- and S1251N-CFTR was similar to normal, the baseline level of chloride transport was b10% of normal CFTR (Figs. 1 and 3; Table 2). Login to comment 69 ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn S549R-CFTR showed maturation comparable to 26±2% (n=5) of normal CFTR (Fig. 1D), which did not account for the b1% of normal CFTR function (Fig. 3; Table 2). Login to comment 70 ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn These results indicated that the function of S549N-, S549R-, and S1251N-CFTR at the cell surface was minimal. Login to comment 71 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg Patch-clamp studies confirmed that the channel open probability of S549N-, S549R-, and S1251N-CFTR was b5% of normal CFTR, whereas the single channel current amplitude Normal F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0 50 100 150 200 CFTRmRNA (%NormalCFTR) None F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0.0 0.2 0.4 0.6 0.8 1.0 ** * CFTRMaturation (Mature/Total) None F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0 100 200 300 400 ** * * * CFTR Mutations MatureCFTR (%NormalCFTR) A B D C Mature Immature Fig. 1. Login to comment 72 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg Patch-clamp studies confirmed that the channel open probability of S549N-, S549R-, and S1251N-CFTR was b5% of normal CFTR, whereas the single channel current amplitude Normal F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0 50 100 150 200 CFTR mRNA (% Normal CFTR) None F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0.0 0.2 0.4 0.6 0.8 1.0 ** * CFTR Maturation (Mature/Total) None F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0 100 200 300 400 ** * * * CFTR Mutations Mature CFTR (% Normal CFTR) A B D C Mature Immature Fig. 1. Login to comment 82 ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn Taken together, these data suggested that defective channel gating was the predominant defect in S549N-, S549R- and S1251N-CFTR. Login to comment 83 ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn In FRT cells expressing S549N-, S549R-, and S1251N-CFTR, acute addition of 10 μM ivacaftor increased the channel open probability and total chloride transport by N10-fold over baseline levels (Figs. 2 and 3; Tables 1 and 2). Login to comment 84 ABCC7 p.Gly551Asp ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn The EC50 for ivacaftor in FRT cells expressing S549N-, S549R-, and S1251N-CFTR were similar to G551D-CFTR (Fig. 3C; Table 2). Login to comment 85 ABCC7 p.Gly551Asp ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn These results indicated that S549N-, S549R-, and S1251N-CFTR were potentiated by ivacaftor in vitro. Login to comment 86 ABCC7 p.Ser1251Asn These results indicated that S549N-, S549R-, and S1251N-CFTR were potentiated by ivacaftor in vitro. Login to comment 89 ABCC7 p.Gly551Asp The most common CF-causing CFTR gating mutation is G551D [6] but several other CFTR gating mutations have been previously reported [9-11]. Login to comment 90 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Gly178Arg In a panel of FRT cells expressing G551D-, G178R-, G551S-, G970R-, G1244E-, S1255P-, and G1349D-CFTR, we confirmed that all these mutant CFTR forms shared similar in vitro functional characteristics that were consistent with a defect in channel gating. Login to comment 91 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Gly178Arg In addition, we showed that the 3 additional mutations, S549N, S549R, and S1251N also have characteristics consistent with gating defects. Login to comment 92 ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn In addition, we showed that the 3 additional mutations, S549N, S549R, and S1251N also have characteristics consistent with gating defects. Login to comment 93 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Gly178Arg Ivacaftor addition caused a N10-fold increase in CFTR-mediated chloride transport in FRT cells expressing G551D-, G178R-, S549N-, S549R-, G551S-, G970R-, G1244E-, S1251N-, S1255P-, and G1349D-CFTR. Login to comment 94 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Gly178Arg Ivacaftor addition caused a N10-fold increase in CFTR-mediated chloride transport in FRT cells expressing G551D-, G178R-, S549N-, S549R-, G551S-, G970R-, G1244E-, S1251N-, S1255P-, and G1349D-CFTR. Login to comment 95 ABCC7 p.Gly551Asp Moreover, in patients with CF who have the G551D CFTR gating mutation, administration of ivacaftor was associated with improvements in CFTR function, as determined by a reduction in sweat chloride concentrations, and improvements in lung function, as determined by an increase in the FEV1 [13]. Login to comment 96 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Taken together, these in vitro results provide a rationale for testing the potential benefit of ivacaftor in individuals with CF who have a CFTR gating mutation other than G551D, including the G178R-, S549N-, S549R-, G551S-, G970R-, G1244E-, S1251N-, S1255P, and G1349D CFTR gating mutations. Login to comment 97 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Evaluation of CF-associated CFTR mutations that were expected to cause protein alterations in the ATP-binding sites formed by the NBDs indicated that S549N- and S1251N-CFTR also shared similar in vitro functional characteristics with G551D-CFTR and could be classified as CFTR gating mutations. Login to comment 98 ABCC7 p.Gly551Asp ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn Unlike S549N-CFTR and S1251N-CFTR, S549R-CFTR exhibited a partial reduction in the level of CFTR maturation compared to normal CFTR (see also Ref. [16]). Login to comment 99 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg The partial reduction in S549R-CFTR maturation was ~27% of A Normal G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 250 Baseline With 10 µM Ivacaftor * * * * * * * * * * * CFTR Mutation ChannelOpenProbability ChannelOpenProbability (%NormalCFTR) B 1pA 3sec + 10 µM Ivacaftor G1349D S1255P G970R G551S G178R G1244E Baseline Normal G551D S1251N S549N S549R Fig. 2. Login to comment 100 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg The partial reduction in S549R-CFTR maturation was ~27% of A Normal G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0.0 0.2 0.4 0.6 0.8 1.0 0 50 100 150 200 250 Baseline With 10 &#b5;M Ivacaftor * * * * * * * * * * * CFTR Mutation Channel Open Probability Channel Open Probability (% Normal CFTR) B 1pA 3sec + 10 &#b5;M Ivacaftor G1349D S1255P G970R G551S G178R G1244E Baseline Normal G551D S1251N S549N S549R Fig. 2. Login to comment 106 ABCC7 p.Ser549Arg Patch-clamp studies showed that the channel open probability of S549R-CFTR was less than 1% of normal CFTR. Login to comment 107 ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg These results indicated that although the predominant defect caused by the S549R CFTR mutation was defective CFTR channel gating, a mild processing defect was also observed. Login to comment 108 ABCC7 p.Ser549Arg These results indicated that although the predominant defect caused by the S549R CFTR mutation was defective CFTR channel gating, a mild processing defect was also observed. Login to comment 110 ABCC7 p.Gly551Asp Patients with CF who carry the G551D CFTR gating mutation have minimal amounts of CFTR function, as determined by high (~100 mmol/L) sweat chloride concentrations, and a severe CF phenotype [8,17]. Login to comment 111 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Although there are limited case studies on patients with CF who have CFTR gating mutations beyond G551D, they also appear to have high sweat chloride concentrations and severe CF [18-20]. Login to comment 112 ABCC7 p.Gly551Asp Although there are limited case studies on patients with CF who have CFTR gating mutations beyond G551D, they also appear to have high sweat chloride concentrations and severe CF [18-20]. Login to comment 113 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser A milder CF clinical phenotype has been associated with the G551S CFTR gating mutation, as demonstrated by a lower sweat chloride concentration (75-94 mmol/L) and lower incidence of pancreatic insufficiency compared to patients with CF who carry the G551D CFTR gating mutation [21]. Login to comment 114 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser In the present study, although the expression and channel open probability of G551S-CFTR were similar to G551D-CFTR, the baseline chloride transport was higher (~9% normal CFTR). Login to comment 115 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser This may be due to the increased level of mature G551S-CFTR delivered to the cell surface compared to normal CFTR, as determined by immunoblot studies in FRT cells. Login to comment 116 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser The potency (EC50) of ivacaftor for the CFTR gating mutations tested was similar to G551D in vitro, suggesting that a similar dose of ivacaftor as that used in clinical trials of patients with CF who carry the G551D CFTR gating mutation may be appropriate for most other CFTR gating mutations. Login to comment 117 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Previous in vitro studies using recombinant cells and primary human airway cultures have shown that higher concentrations of ivacaftor were required to potentiate G551D-CFTR compared to normal- and F508del-CFTR [12]. Login to comment 118 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp The potency of other CFTR potentiators has also been shown to be lower for G551D-CFTR compared to normal or F508del-CFTR [12,22-25]. Login to comment 119 ABCC7 p.Gly551Asp The potency of other CFTR potentiators has also been shown to be lower for G551D-CFTR compared to normal or F508del-CFTR [12,22-25]. Login to comment 122 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly970Arg These included derivatives of 1,4-dihydropyridine and phenylglycine which potentiated G551D-, G970R-, and G1349D-CFTR to a similar extent as ivacaftor [23,25-26]. Login to comment 123 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg In contrast, sulfamoyl-4-oxoquinoline-3-carboxamides were weakly effective on G551D-, G970R-, and G1349D-CFTR [26] and phloxine B strongly potentiated G551D-CFTR, but not G1349D-CFTR [22]. Login to comment 124 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly970Arg In contrast, sulfamoyl-4-oxoquinoline-3-carboxamides were weakly effective on G551D-, G970R-, and G1349D-CFTR [26] and phloxine B strongly potentiated G551D-CFTR, but not G1349D-CFTR [22]. Login to comment 127 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn Like G551D, the G551S, G1244E, S1255P, and G1349D CFTR gating mutations, as well as the S549N, S549R, and S1251N CFTR gating mutations identified in the Table 1 Effect of ivacaftor on the channel gating activity of CFTR with gating mutations. Login to comment 128 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Single channel current amplitude at 80 mV CFTR channel open probability Baseline With 10 bc;M ivacaftor Baseline With 10 μM ivacaftor Mutation pA % Normal pA % Normal Po % Normal Po % Normal Normal 0.57±0.03 100 0.63±0.02 111 0.400±0.04 100 0.800±0.04 a 200 G551D 0.46±0.06 81 0.46±0.03 81 0.019±0.01 b 5 0.121±0.035 a 30 G178R 0.59±0.11 103 0.66±0.08 116 0.005±0.001 b 1 0.228±0.022 a 57 S549N 0.55±0.02 97 0.61±0.02 108 0.003±0.010 b 1 0.396±0.119 a 99 S549R 0.45±0.01 b 79 0.55±0.02 a 96 0.004±0.010 b 1 0.143±0.031 a 36 G551S 0.57±0.13 100 0.64±0.02 113 0.010±0.001 b 3 0.337±0.110 a 84 G970R 0.55±0.03 96 0.55±0.03 97 0.001±0.001 b 0 0.245±0.042 a 61 G1244E 0.44±0.11 77 0.54±0.08 94 0.011±0.010 b 3 0.470±0.122 a 118 S1251N 0.54±0.07 95 0.63±0.04 111 0.003±0.010 b 1 0.350±0.03 a 88 S1255P 0.70±0.03 b 122 0.71±0.02 125 0.018±0.016 b 5 0.468±0.168 a 117 G1349D 0.49±0.08 85 0.63±0.06 111 0.019±0.015 b 5 0.315±0.110 a 79 a Significantly different (Pb0.05; paired t-test, n=3-5) compared to baseline levels for each CFTR mutation. Login to comment 129 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg Single channel current amplitude at 80 mV CFTR channel open probability Baseline With 10 bc;M ivacaftor Baseline With 10 bc;M ivacaftor Mutation pA % Normal pA % Normal Po % Normal Po % Normal Normal 0.57&#b1;0.03 100 0.63&#b1;0.02 111 0.400&#b1;0.04 100 0.800&#b1;0.04 a 200 G551D 0.46&#b1;0.06 81 0.46&#b1;0.03 81 0.019&#b1;0.01 b 5 0.121&#b1;0.035 a 30 G178R 0.59&#b1;0.11 103 0.66&#b1;0.08 116 0.005&#b1;0.001 b 1 0.228&#b1;0.022 a 57 S549N 0.55&#b1;0.02 97 0.61&#b1;0.02 108 0.003&#b1;0.010 b 1 0.396&#b1;0.119 a 99 S549R 0.45&#b1;0.01 b 79 0.55&#b1;0.02 a 96 0.004&#b1;0.010 b 1 0.143&#b1;0.031 a 36 G551S 0.57&#b1;0.13 100 0.64&#b1;0.02 113 0.010&#b1;0.001 b 3 0.337&#b1;0.110 a 84 G970R 0.55&#b1;0.03 96 0.55&#b1;0.03 97 0.001&#b1;0.001 b 0 0.245&#b1;0.042 a 61 G1244E 0.44&#b1;0.11 77 0.54&#b1;0.08 94 0.011&#b1;0.010 b 3 0.470&#b1;0.122 a 118 S1251N 0.54&#b1;0.07 95 0.63&#b1;0.04 111 0.003&#b1;0.010 b 1 0.350&#b1;0.03 a 88 S1255P 0.70&#b1;0.03 b 122 0.71&#b1;0.02 125 0.018&#b1;0.016 b 5 0.468&#b1;0.168 a 117 G1349D 0.49&#b1;0.08 85 0.63&#b1;0.06 111 0.019&#b1;0.015 b 5 0.315&#b1;0.110 a 79 a Significantly different (Pb0.05; paired t-test, n=3-5) compared to baseline levels for each CFTR mutation. Login to comment 130 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg 0 100 200 300 400 -9 -8 -7 -6 -5 -4 G178R G551D G551S 0 S549N S549R Ivacaftor [Log M] 0 100 200 300 400 0 50 100 150 200 -9 -8 -7 -6 -5 -4 G970R G1244E S1255P G1349D 0 S1251N Ivacaftor [Log M] ChlorideTransport (%NormalCFTR) Normal Forskolin G178R G551S G970R G1244E 50 2 1 min S1255P Normal F508del G551D G178R S549N S549R G551S G970R G1244E S1251N S1255P G1349D 0 100 200 300 400 0 50 100 150 200 * * * * * * * * * * * * * CFTR Mutation ChlorideTransport(µA/cm2)ChlorideTransport(µA/cm2) ChlorideTransport(A/cm2) ChlorideTransport (%NormalCFTR) B G1349D G551D A F508del C S549N S549R S1251N Baseline Baseline present study, cause protein alterations in the ATP binding pockets formed by the two NBDs required for normal CFTR channel gating (Fig. 4) [2]. Login to comment 131 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg ABCC7 p.Gly178Arg The G178R and G970R CFTR gating mutations alter the intracellular cytoplasmic loops that are believed to link the ATP-driven conformational changes in the NBDs to the opening of the CFTR channel pore formed by the membrane spanning domains [27]. Login to comment 132 ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg The G178R and G970R CFTR gating mutations alter the intracellular cytoplasmic loops that are believed to link the ATP-driven conformational changes in the NBDs to the opening of the CFTR channel pore formed by the membrane spanning domains [27]. Login to comment 138 ABCC7 p.Arg117His For example, R117H-CFTR is delivered to the cell surface in normal amounts, but exhibits a ~20% reduction in CFTR channel conductance and a ~75% reduction in the channel open probability, resulting in residual CFTR function both in vitro and clinically [28]. Login to comment 139 ABCC7 p.Arg117His ABCC7 p.Arg347His ABCC7 p.Asp1152His Other CFTR gene mutations associated with residual CFTR function include A445E, R347H, D1152H, and certain splice mutations (3849 +10kbC→T) [4,29-30]. Login to comment 140 ABCC7 p.Arg347His ABCC7 p.Asp1152His Other CFTR gene mutations associated with residual CFTR function include A445E, R347H, D1152H, and certain splice mutations (3849 +10kbC࢐T) [4,29-30]. Login to comment 142 ABCC7 p.Gly551Asp This suggested that ivacaftor is not a mutation-specific CFTR potentiator, as it overcomes the underlying molecular defect caused by G551D and a number of other CFTR gating mutations. Login to comment 143 ABCC7 p.Gly551Asp This suggested that ivacaftor is not a mutation-specific CFTR potentiator, as it overcomes the underlying molecular defect caused by G551D and a number of other CFTR gating mutations. Login to comment 144 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg The in vitro data presented here suggest that ivacaftor has a similar effect on all CFTR forms with gating defects and support the investigation of ivacaftor in patients with CF who have CFTR gating mutations beyond G551D, including G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P, and G1349D. Login to comment 145 ABCC7 p.Gly551Asp ABCC7 p.Gly551Ser ABCC7 p.Gly1244Glu ABCC7 p.Gly1349Asp ABCC7 p.Ser1255Pro ABCC7 p.Ser1251Asn ABCC7 p.Ser549Arg ABCC7 p.Ser549Asn ABCC7 p.Gly970Arg ABCC7 p.Gly178Arg The in vitro data presented here suggest that ivacaftor has a similar effect on all CFTR forms with gating defects and support the investigation of ivacaftor in patients with CF who have CFTR gating mutations beyond G551D, including G178R, S549N, S549R, G551S, G970R, G1244E, S1251N, S1255P, and G1349D. Login to comment