ABCMdb

ABCC7 p.Val232Asp

II-III (maturation defect, gating defect) <a href="https://www.ncbi.nlm.nih.gov/pubmed/26823392">Veit et al.</a>
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Publications

PMID: 10794365 [PubMed] Bernardino AL et al: "Molecular analysis in Brazilian cystic fibrosis patients reveals five novel mutations."

No. Sentence Comment

6 Another fifteen mutations (previously reported) were detected: G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 6211 1GRT, V232D, 1717-1GRA, 2347 delG, R851L, 27891 5GRA, and W1089X. Login to comment
53 Seven other rare mutations were also identified : 6211 1GRT (exon 4), V232D (exon 6a), 1717-1G RA (intron 11), 2347 delG (exon 13b), R851L (exon 14a), 27891 5GRA (intron 14b), and W1089X (exon 17b). Login to comment
81 In this study, 16 mutations were identified: D F508, G542X, R1162X, N1303K, R334W, W1282X, G58E, L206W, R553X, 6211 1GRT, V232D, 1717-1GRA, 2347 delG, R851L, 27891 5GRA, and W1089X. Login to comment
84 GEN OTYPES, FREQUENCIES, AN D PRESENCE OF PI FRO M 160 CF PATIE NTS (320 CF CHROM OSOM ES) Number and frequency (%) Genotype Number Frequency (%) of patients with PI D F508/D F508 47 29.40 47 (100%) D F508/G542X 13 8.10 13 (100%) D F508/R1162X 6 3.80 6 (100%) D F508/R334W 5 3.10 3 (60%) D F508/N1303K 3 1.90 3 (100%) D F508/W1282X 2 1.20 2 (100%) D F508/G58E 2 1.20 1 (50%) D F508/L206W 1 0.62 0 D F508/R553X 1 0.62 1 (100%) D F508/R851L 1 0.62 0 D F508/2789 1 5g ® A 1 0.62 0 D F508/3617delGA 1 0.62 1 (100%) D F508/3171delC 1 0.62 1 (100%) D F508/2686insT 1 0.62 1 (100%) D F508/Y275X 1 0.62 1 (100%) D F508/U 22 13.80 14 (64%) G542X/G542X 3 1.90 3 (100%) G542X/N1303K 3 1.90 2 (67%) G542X/R1162X 1 0.62 1 (100%) G542X/U 5 3.10 4 (80%) N1303K/R1162X 1 0.62 1 (100%) N1303K/G58E 1 0.62 0 2347delG/2347delG 1 0.62 1 (100%) R334W/V232D 1 0.62 0 R334W/W1089X 1 0.62 1 (100%) R334W/U 1 0.62 1 (100%) W1282X/U 1 0.62 1 (100%) G58E/U 1 0.62 1 (100%) R553X/U 1 0.62 1 (100%) L206W/U 1 0.62 0 621 1 1G ® T/U 1 0.62 1 (100%) 1717-1G ® A/U 1 0.62 Not known V201M/U 1 0.62 0 U/U 27 16.90 12 (44%) Total 160 100 - U, Unknown CF mutation. Login to comment

PMID: 10875853 [PubMed] Casals T et al: "Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens."

No. Sentence Comment

73 Finally, six polymorphisms were found each in V232D 2 (1) 0 2 (1) one patient: 104G/T, 296ϩ128G/C, 741C/T, 3195A/T, 3212T/3732delA 0 2 (14) 2 (1) L383S 1 1 (7) 2 (1) C and 4029A/G. Login to comment
83 We detected only three homozygous patients (one for V232D P Ͻ 0.001). Login to comment
97 Dilatation V232D/V232D 9T/9T 1 of ejaculatory ducts, often resembling utricular cysts, was S945L/R258G 7T/7T 1 demonstrable also in some men, all of whom were azoospermicG551D/F1074L 5T/7T 1 A1006E/L383S 5T/7T 1 (Figure 1). Login to comment

PMID: 11466205 [PubMed] Larriba S et al: "Adenosine triphosphate-binding cassette superfamily transporter gene expression in severe male infertility."

No. Sentence Comment

87 Phenotypical and genotypical description of CAVD and non-CAVD infertile patients.a No. patient Phenotype FSH (U/L) Non-CFTR infertility-associated factors Testicular biopsy CFTR mutation M470V polymorphism CAVD infertility 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CBAVD CUAVD CUAVD CUAVD CUAVD 3.1 7.3 3.1 2.4 1.9 3.5 5.7 4.3 3.6 ND 2.2 4.8 11.3 2.1 ND 7.6 5.3 6.5 3.9 21.4 None None None None None None None None None None None None None None None None None None None Yes 1 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes V232D/V232D F508del/R117H F508del/R117H G542X/2789ϩ5GϾA F508del/D1270N ϩ R74W F508del/D1270N ϩ R74W S945L/R258G F508del/5T F508del/5T L206W/5T R117H/N F508del/N Y1014C/N 5T/N N/N N/N Y1092X/R258G 621ϩ1GϾT/5T Q890R/N N/N M/M M/M M/M M/M M/V M/V M/V M/M M/V M/V M/V M/V M/V M/V M/M V/V V/V M/V V/V M/M Non-CAVD infertility 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SA) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SSO) TF (SA) TF (SA) TF (SSO) OA OA OA OA OA OA OA OA 42.0 15.9 34.8 8.9 26.3 6.4 7.8 15.6 8.7 3.2 3.9 12.6 4.7 1.3 5.6 3.9 6.1 9.3 8.8 19.3 9.6 ND 3.3 5.9 6.6 3.6 1.9 4.2 2.0 4.4 None None None None None None None None None None None None None None None None Yes 2 Yes 2 Yes 2, 3 Yes 4 Yes 5 Yes 6 None None None None None Yes 1 Yes 7 Yes 8 Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes F508del/N R334W/N N/N N/N N/N N/N N/N N/N N/N R75Q/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N 5T/5T N/N N/N N/N N/N N/N N/N N/N M/M V/V M/V M/V M/V M/V V/V V/V V/V V/V M/V M/V M/V ND V/V M/M M/V M/M M/V M/M M/V V/V M/V M/V M/V V/V V/V M/V M/V V/V a CFTR mutations and M470V allele are also described for each patient. Login to comment
94 CFTR Analysis We have identified 14 different CFTR mutations (R117H, L206W, V232D, R258G, F508del, G542X, 621ϩ1GϾT, Q890R, S945L, Y1014C, Y1092X, D1270N, 2789ϩ5GϾA, IVS8-6[5T]) in 17 of 20 patients of the CAVD group, giving a CFTR mutation frequency of 85%. Login to comment

PMID: 11748233 [PubMed] Therien AG et al: "Interhelical packing in detergent micelles. Folding of a cystic fibrosis transmembrane conductance regulator construct."

No. Sentence Comment

136 dition, since TM 3-4 constructs represent a segment of the CFTR protein, experiments become feasible which test the effects of various cystic fibrosis phenotypic mutations on interhelical packing of TMs 3 and 4, as described for the mutant V232D (21). Login to comment

PMID: 11781704 [PubMed] Larriba S et al: "ATB(0)/SLC1A5 gene. Fine localisation and exclusion of association with the intestinal phenotype of cystic fibrosis."

No. Sentence Comment

151 Statistical analysis showed that the higher incidence for P17A and the lower incidence for V512L observed in the general population Table 3 CFTR mutations of the CF patients under study with and without meconium ileus (MI) CF-non MI CF-MI CFTR mutations n CFTR mutations n F508del/R117H 2 F508del/F508del 7 F508del/R334W 3 F508del/L365P 1 F508del/R347P 1 F508del/G542X 1 F508del/621+1G4Ta 1 F508del/621+IG4Ta 1 F508del/M1101K 1 F508del/R1066C 1 F508del/1609delCAa 1 F508del/W1089X 1 F508del/2789+5G4Aa 3 F508del/R1162X 1 F508del/3849+10kbC4T 1 F508del/1609delCAa 1 G542X/G85E 1 F508del/Q1281X 1 G542X/V232D 1 F508del/1811+1.6kbA4G 1 G542X/1811+1.6kb A4Ga 1 F508del/2789+5G4Aa 1 G542X/2789+5G4A 1 F508del/2869insG 1 Q890X/L206W 1 F508del/unknown 1 1811+1.6kbA4G/P205S 1 I507del/I507del 1 R1162X/3272-26A4G 1 G542X/1078delT 1 N1303K/R347H 1 G542X/1811+1.6kbA4Ga 1 N1303K/A1006E+5T 1 S549R/CFTR50kbdel 1 2789+5G4A/405+1G4A 1 R1066C/R1066C 1 W1282X/712-1G4T 1 a CF patient with a sibling presenting identical CFTR genotype and discordance of intestinal phenotype. Login to comment

PMID: 11888281 [PubMed] Partridge AW et al: "Polar residues in membrane domains of proteins: molecular basis for helix-helix association in a mutant CFTR transmembrane segment."

No. Sentence Comment

1 To systematically explore the structural consequences of H-bonds between TM helices, we focused on TM4 of the cystic fibrosis conductance regulator (CFTR) and its cystic fibrosis- (CF-) phenotypic mutation, V232D, as a model system. Login to comment
2 Synthetic peptides corresponding to wild-type (TM4-wt) (residues 219-242: LQASAFCGLGFLIVLALFQAGLGR) and mutant (TM4-V232D) sequences both adopt helical structures in SDS micelles and display dimer bands on SDS-PAGE arising from disulfide bond formation via wild-type residue Cys-225. Login to comment
3 However, the TM4-V232D peptide additionally forms a ladder of noncovalent oligomers, including tetramers, hexamers, and octamers, mediated by a hydrogen bond network involving Asp-Gln side chain-side chain interactions. Login to comment
19 To investigate the roles of polar side chains in TM helices, we synthesized peptides corresponding to TM4 from CFTR either with or without the CF V232D mutation. Login to comment
20 We found that whereas the wild-type TM4 peptide failed to form any noncovalent helical associations, the TM4 peptide containing the V232D mutation generates a well-defined oligomeric ladder of TM4 helices mediated through a network of interhelical side chain-side chain H-bonds between D232 † This work was supported, in part, by grants to C.M.D. from the Canadian Cystic Fibrosis Foundation, the Canadian Institutes for Health Research (CIHR), and the National Institutes of Health (NIDDK). Login to comment
65 The TM4 peptide was designed identically to TM4-wt except that it contained the V232D mutation. Login to comment
67 Effect of the V232D Mutation on Helical Packing. Login to comment
71 However, the TM4 peptide containing the V232D mutation displays additional bands consistent with those of a tetramer, hexamer, and octamer. Login to comment
122 In this context, we simulated H-bond formation in membrane-interactive peptides using segments featuring the CF-phenotypic mutation V232D from TM4 of the CFTR transmembrane domain. Login to comment
135 The V232D peptide was reduced in the lane indicated by incubating the peptide with TCEP in aqueous solution for 5 min prior to addition of Tricine sample buffer. Login to comment
163 However, due to FIGURE 6: Molecular models proposed for the oligomerization of the CFTR transmembrane helix 4 containing the V232D mutation (TM4-VD). Login to comment
178 The V232D mutation in TM4 creates a membrane-buried electrostatic locus, whether it is in the context of a TM peptide or in the full-length protein. Login to comment
182 For example, where the V232D mutation arises in full-length CFTR, the D232 residue on one TM4 helix could partner with the Q237 residue in the complementary TM4 helix in another CFTR molecule, to produce a functionally impaired H-bonded dimer. Login to comment
185 In the single-spanning TM peptides studied here, the V232D mutation results in a homophilic association of TM4 helices mediated by D232 and Q237 interhelical H-bonding. Login to comment
187 That the V232D mutation produces a non-wild-type H-bond in both systems suggests that a given mutant polar residue arising in the CFTR membrane domain could have multiple polar partners available in vivo. Login to comment

PMID: 15204624 [PubMed] Booth PJ et al: "Polytopic membrane protein folding and assembly in vitro and in vivo."

No. Sentence Comment

88 In the case of cystic fibrosis (see also below), detailed studies of peptides corresponding to helixÁ/ loopÁ/helix segments of the CFTR protein have shown that a hydrophobic-to-charged point mutation (V232D) in TM helix 4 induces a hydrogen bond with Gln 207 in helix 3, where this alteration is known to result in a cystic fibrosis disease phenotype [37]. Login to comment

PMID: 15776432 [PubMed] Clain J et al: "Misprocessing of the CFTR protein leads to mild cystic fibrosis phenotype."

No. Sentence Comment

256 This hypothetical disease-causing mechanism is based on the observation that the neutral-to-charge CF mutation p.V232D in TM4 induces an aberrant side chain-side chain H bond with the neighboring wild-type Gln-207 in TM3 by using a recombinant helix-loop-helix fragment of CFTR (Therien et al., 2001). Login to comment

PMID: 16980811 [PubMed] Kammesheidt A et al: "Comprehensive genetic analysis of the cystic fibrosis transmembrane conductance regulator from dried blood specimens--implications for newborn screening."

No. Sentence Comment

98 In states with single specimenmodels,originalspecimensaretestedforthepresenceof themostcommonmutation,deltaF508,and/orotherdeleterious Table 1 Genotype data from panel testing and comprehensive Ambry TestTM : CF analysis Case Ethnicity ABI-31 Mutation 1 ABI-31 Mutation 2 Genzyme-87 Mutation 1 Genzyme-87 Mutation 2 Ambry Mutation 1 Ambry Mutation 2 Ambry Mutation 3 1 Hispanic delF508a 4382delAa 2 Hispanic delF508 N/I delF508 N/I delF508a 1248ϩ1GϾAa 3 African-American N/I N/I N/I N/I M150K CFTRdele17A,17Bb 4 Hispanic G542X N/I G542X N/I G542Xa 1288insTAa 5 African-American N/I N/I 3120ϩ1GϾA N/I 3120ϩ1GϾAa Q98Xa 3849؉72G>A 6 Hispanic delF508 N/I delF508 N/I delF508a 2289del10ins5a 7c Hispanic N/I N/I N/I N/I H199Ya 406-1GϾAa 8 Hispanic delF508 N/I delF508 N/I delF508a CFTRdele2,3(21kbb 9 Hispanic delF508 N/I delF508 N/I delF508a 2105-2117del13insAGAAAa 10 Hispanic G542X N/I G542X N/I G542X M952I Y914X 11 Hispanic N/I N/I N/I N/I 663delT L558S 12 Hispanic N/I N/I delF311 N/I delF311a 406-1GϾAa 13 Hispanic N/I N/I 2055del9insAa 2055del9insAa 14 Hispanic delF508 N/I delF508 N/I delF508 2055del9insA 15 Hispanic delF508 N/I delF508 N/I delF508 E257X 16 Hispanic N/I N/I N/I N/I V232D V232D 17 Hispanic delF508 N/I delF508 N/I delF508 H199Y 18 Hispanic delF508 N/I delF508 4160insGGGG 19 Caucasian delF508 N/I delF508 297-1GϾA 20 Hispanic 2183delAAϾG N/I 2183delAAϾG N/I 2183de1AAϾG 3500-2AϾG 21 Hispanic delF508 N/I delF508 S492F 22 Hispanic delF508 N/I delF508 N/I delF508 935delA 23 Caucasian R1162X N/I R1162X N/I R1162X 3940delG 24 Hispanic 711ϩ1GϾT N/I 711ϩ1GϾT T465N 25 Hispanic delF508 N/I delF508 N/I delF508 406-1GϾA 26 Hispanic delF508 N/I delF508 2055del9insA 27 Hispanic delF508 N/I delF508 N/I delF508 V232D 28 Hispanic delF508 N/I delF508 N/I delF508 S1235R 29 Hispanic G542X N/I G542X N/I G542X 297-1GϾA 30 Hispanic delF508 N/I delF508 N/I delF508 Q1100P 31 Hispanic delF508 N/I delF508 W216X 32 Hispanic N/I N/I N/I N/I 406-1GϾA H199Y 33 Hispanic N/I N/I N/I N/I 3272-26AϾG R75X 34 Hispanic N/I N/I Q890X N/I Q890X 2055del9insA 35 Hispanic delF508 N/I delF508 N/I delF508 W216X 36 Hispanic delF508 N/I delF508 N/I delF508 H199Y 37 Hispanic delF508 N/I delF508 N/I delF508 1288insTA I1027T 38 Hispanic G542X N/I G542X N/I G542X 663delT 39 Hispanic delF508 N/I delF508 N/I delF508 1288insTA 40 Hispanic delF508 N/I delF508 1288insTA mutations using mutation panels. Login to comment

PMID: 17329263 [PubMed] Ratbi I et al: "Detection of cystic fibrosis transmembrane conductance regulator (CFTR) gene rearrangements enriches the mutation spectrum in congenital bilateral absence of the vas deferens and impacts on genetic counselling."

No. Sentence Comment

93 1 Two CFTR mutations 15 0-15 0 [R117H] þ [(TG)13(T)5] 1 [R117H] þ [(TG)12(T)5] 1 [R117H] þ [(TG)11(T)5] 1 [R117H] þ [M952I] 1 [D1152H] þ [(TG)12(T)5] 2 [D1152H] þ [Y1032C] 1 [(TG)11(T)5;V562I] þ [L997F] 1 [(TG)11(T)5;V562I] þ [S977F] 1 [E1473X] þ [(TG)13(T)5] 1 [V232D] þ [(TG)12(T)5] 1 [R334W] þ [(TG)12(T)5] 1 [G622D] þ [(TG)12(T)5] 1 [3272-26A . Login to comment

PMID: 17516627 [PubMed] Wehbi H et al: "Role of the extracellular loop in the folding of a CFTR transmembrane helical hairpin."

No. Sentence Comment

44 Briefly, the percent change in molecular weight (MW) was first calculated from gels by comparing the apparent MW of each TM3/4 hairpin [estimated from the migration rates of Mark-12 molecular weight markers (Invitrogen)] to its expected theoretical MW using the equation: The percent change in MW of each ECL2 replacement in the TM3/4 WT or TM3/4 V232D backgrounds was then normalized to the migration of the parental hairpin as follows: ECL2 mutants with positive percent changes thus migrate less than their parental hairpin on SDS-PAGE; those with negative values migrate further. Login to comment
97 When the changes in TM3/4 WT hairpin migration were compared to changes in overall hairpin helicity, a strong correlation (R ) 0.79) was observed (Figure 5), leading us to propose that increases in non-native R-helix structure within ECL2 might Table 1: Migration Behavior on SDS-PAGE Gels of Single and Double Mutants in the Loop Region of CFTR TM3/4 Constructs % change in apparent MW on SDS-PAGE mutant vs TM3/4 WT in WT loop mutantsa vs TM3/4 V232D in V232D loop mutantsa Pb E217G 6.8 ( 0.7 E217S 11.1 ( 3.4 5.4 ( 1.4 0.056 Q220R 15.2 ( 1.1 Q220G 0.3 ( 0.4 Q220N 2.1 ( 1.3 0.5 ( 0.3 0.108 Q220K 14.1 ( 1.0 Q220W 13.1 ( 1.3 11.5 ( 0.9 0.157 Q220E -11.1 ( 1.1 -4.0 ( 0.3 <0.001 S222G 12.0 ( 2.1 1.1 ( 0.6 0.001 S222E -0.3 ( 2.4 1.3 ( 0.5 0.512 E217G/S222G 12.4 ( 1.9 E217S/S222E 26.1 ( 4.5 averagec 10.4 ( 7.3 4.0 ( 4.2 0.067 a Values are the percentage difference vs TM3/4 WT or TM3/4 V232D migration of SDS-PAGE gels. Login to comment
112 ECL2 Mutants Are Less DisruptiVe in the Context of the Membrane-Based CF-Phenotypic TM Mutant V232D. Login to comment
114 The CF-phenotypic V232D replacement in TM helix 4 has been suggested in the TM3/4 hairpin context to form a stabilizing interhelical hydrogen bond (33, 38); the TM3/4 V232D construct has a more compact fold than the TM3/4 WT hairpin and consequently migrates faster on SDS-PAGE (33). Login to comment
115 Several ECL2 replacements characterized as described above in the TM3/4 WT background were recapitulated in the TM3/4 V232D hairpin (Figure 6, Table 1). Login to comment
116 We found that the Q220E, S222G, and E217S replacements exhibited a relatively less pronounced effect on migration in the TM3/4 V232D hairpin than the TM3/4 WT background with high (p < 0.01 for Q220E and S222G) or detectable but marginal (p ) 0.056 for E217S) statistical significance. Login to comment
117 The Q220N and Q220W replacements also affected folding less in the TM3/4 V232D background than in the TM3/4 WT hairpin (Table 1). Login to comment
118 The mean change in migration of ECL2 mutants in the TM3/4 V232D hairpin was >2-fold smaller than the WT. Login to comment
139 Several sets of experi- FIGURE 6: SDS-PAGE migration of S222G and Q220W replacements in the TM3/4 WT and TM3/4 V232D backgrounds. Login to comment
140 Hairpins containing ECL2 mutants S222G and Q220W in the TM3/4 WT background and in the TM3/4 V232D background are shown. Login to comment
143 ments suggest that TM3/4 migration patterns are not simple functions of charge: (i) The double mutant Q207L/V232D migrates identically to WT, while V232D migrates significantly faster than WT (33). Login to comment
160 The TM3/4 V232D construct exhibits marginal but discernibly smaller migration changes than TM3/4 WT when ECL2 substitutions are introduced (Table 1, Figure 6). Login to comment
161 The stronger helix-helix interactions in TM3/4 V232D may therefore help to maintain a compact hairpin structure when ECL2 loop mutants are introduced that otherwise destabilize the fold. Login to comment

PMID: 18193900 [PubMed] Cheung JC et al: "Misfolding of the cystic fibrosis transmembrane conductance regulator and disease."

No. Sentence Comment

121 V232D is the CF-phenotypic mutant in TM4. Login to comment
141 We addressed the possibility experimentally that wild type Q207 in TM3 can form an interhelical side chain-side chain H-bond with CF-phenotypic mutant V232D in TM4 in an investigation of a series of helix-loop-helix ("hairpin") constructs derived from CFTR TM helices 3 and 4. Login to comment

PMID: 19181854 [PubMed] Rath A et al: "Detergent binding explains anomalous SDS-PAGE migration of membrane proteins."

No. Sentence Comment

5 The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. Login to comment
24 We find that gel shifts strongly correlate (R2 ϭ 0.8) with changes in the SDS-loading capacity of these miniature membrane proteins, indicating that altered detergent binding explains anomalous SDS-PAGE behavior. Our results reveal a distinction between two CF-phenotypic mutants studied: V232D binds significantly less SDS than the WT protein while P205S SDS binding is indistinguishable from WT, indicating that CFTR dysfunction may arise variously as a consequence of altered protein-lipid interactions or via altered intra-protein contacts. Login to comment
61 PA/VD and ES/SE denote the P205A/V232D and E217S/S222E hairpins, respectively. Login to comment
62 V232D, V232A, P205S, and Q220W) migrated as WT within statistical significance; 2 were faster (V232D and V232K); and 4 were slower (G228L, E217V, E217F, and E217S/S222E). Login to comment
70 The mutant hairpins ranged in loading levels from 3.4-10 g SDS/g, with V232D binding significantly fewer SDS molecules than WT, and the E217F and E217S/S222E mutants binding significantly more. Login to comment
97 PA/VD and ES/SE denote the P205A/V232D and E217S/S222E hairpins, respectively. Login to comment
113 Conversely, even though it migrates faster than WT on SDS-PAGE, the V232D hairpin reports a radius approximately 20% larger than WT (Table 2). Login to comment
123 However, even if SDS/ protein stoichiometry (and by extension, gel shift) remains unchanged, increases in the conformational flexibility of non-coated regions may alter the hairpin`s hydrodynamic radius (compare Fig. 5 B-E)-a potential explanation for the as-WT gel shift but increased hydrodynamic radius relative to WT of the P205A/V232D mutant. Login to comment
141 For example, V232D and P205A/V232D display larger than WT hydrodynamic radii on SEC-HPLC (ϩ19% and ϩ21%, respectively)-even though each Asp-containing mutant migrates faster or as-WT on PAGE. Login to comment
153 Our observation that the V232D mutant binds less SDS than WT TM3/4 may therefore indicate that the dysfunction in the full-length CFTR molecule caused by this mutation arises from altered protein-lipid associations. Login to comment

PMID: 19625452 [PubMed] Grove DE et al: "Mechanisms for rescue of correctable folding defects in CFTRDelta F508."

No. Sentence Comment

10 In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Login to comment
183 Thus, we asked to what extent can disease related folding defects caused by mutations in MSD1 (G85E, G91R, and V232D), MSD2 (M1137R), and NBD2 (N1303K) be corrected relative to those caused by deletion of F508 in NBD1 (Figure 4A). Login to comment
185 The V232D mutation has been proposed to introduce an abnormal hydrogen bond within the transmembrane (TM) region of MSD1, which impedes normal TM assembly (Therien et al., 2001), but the affect of this mutation on CFTR biogenesis is unknown. Login to comment
189 The CFTR G85E and G91R point mutations are contained within TM1, whereas the V232D mutation lies within TM4 of CFTR`s MSD1 domain. Login to comment
194 Intriguingly, introduction of the V232D mutation generates an unstable CFTR biogenic mutant with decreased levels of the immature B-form and no apparent maturation product (Figure 4A). Login to comment
195 Yet, treatment with Corr-4a resulted in a dramatic increase in accumulation of both the immature and mature forms of CFTR V232D. Login to comment
196 However, folding defects in CFTR V232D were not significantly rescued by low temperature incubations (data not shown). Login to comment
197 Pulse-chase analysis indicated that Corr-4a strongly increases the folding efficiency of CFTR V232D, and does not simply increase the synthesis of this point mutant (Figure 4B). Login to comment
198 The ability of Corr-4a to dramatically enhance CFTR V232D folding suggests that this mutant may have a single folding defect in MSD assembly that is correctable by Corr-4a. Login to comment
199 In agreement with this idea, Corr-4a is able to enhance the accumulation of the V232D-containing MSD1 fragment, CFTR 370X V232D (data not shown). Login to comment
209 (B) Corr-4a increases the biosynthetic maturation of CFTR V232D. Login to comment
210 HEK293 cells transfected with CFTR V232D (1 ␮g) were preincubated with Corr-4a or DMSO for 2 h, labeled with [35 S]methionine, and chased for the indicated amounts of time in the continuous presence of chemical treatment. Login to comment
227 Next, we probed the ability of Corr-4a to enhance the proper association of CFTR 837X V232D with CFTR 837-1480. Login to comment
228 We observed that, when compared with the wild-type 837X fragment, the steady-state levels of CFTR 837X V232D were decreased (Figure 5B). Login to comment
229 Yet, the levels of CFTR 837X V232D were increased upon Corr-4a treatment. Login to comment
230 Furthermore, pulse-chase analysis demonstrated that Corr-4a stabilizes CFTR 837X V232D (Figure 5C). Login to comment
231 In addition, the levels of the C-terminal fragment, 837-1480, were increased when coexpressed with the CFTR 837X V232D mutant fragment (Figure 5D). Login to comment
233 Yet, when Corr-4a was present, a large subpopulation of the 837X V232D and 837- Figure 5. Login to comment
238 (B) HEK293 cells transfected individually with CFTR 837X (1 ␮g), CFTR 837X⌬F508 (1 ␮g), CFTR 837X V232D (1 ␮g), or CFTR 837-1480 (1 ␮g) were cultured for 18 h before addition of Corr-4a or DMSO. Login to comment
242 (C) Corr-4a increases the stability of CFTR 837X V232D and CFTR 837-1480. Login to comment
243 HEK293 cells transfected with either CFTR 837X V232D or CFTR 837-1480 were preincubated with Corr-4a or DMSO for 2 h, labeled with [35 S]methionine, and chased for the indicated amounts of time in the continuous presence of chemical treatment. Login to comment
247 (D) HEK293 cells were transfected with either CFTR 837-1480, CFTR 837X and CFTR 837-1480, CFTR 837X⌬F508 and CFTR 837-1480, or CFTR 837X V232D and CFTR 837-1480. Login to comment
254 Thus, Corr-4a is able to correct defects in MSD1 folding to the degree that it facilitates the proper assembly of CFTR 837X V232D and CFTR 837-1480. Login to comment
257 Yet, it appears that multiple folding defects limit the rescue of CFTR⌬F508, whereas a less complex mutant, such as CFTR V232D, is readily rescued. Login to comment
309 Corr-4a is also able to act on CFTR 837X V232D, and enhance its accumulation. Login to comment
318 Surprisingly, there was a dramatic enhancement of CFTR V232D folding by Corr-4a. Login to comment
319 In addition, Corr-4a was able to enhance the accumulation of CFTR 837X V232D and to increase levels of the mature, highly glycosylated form of CFTR 837-1480 with CFTR 837X V232D. Login to comment
320 The V232D mutant presents a mild form of CF (Alonso et al., 2007); however, the molecular basis for CF associated with this mutation has not been well-defined. Login to comment
321 The V232D mutation is proposed to form an aberrant hydrogen bond in MSD1 (Therien et al., 2001; Choi et al., 2004) that may hinder formation of interdomain contacts between MSD1 and MSD2 that are proposed to occur in the folded CFTR channel (Dawson and Locher, 2006; Serohijos et al., 2008). Login to comment
325 Yet, rare mutations, such as V232D, which cause more specific folding defects (Therien et al., 2001; Choi et al., 2004) are easier to correct. Login to comment

PMID: 20100616 [PubMed] Havasi V et al: "Association of cystic fibrosis genetic modifiers with congenital bilateral absence of the vas deferens."

No. Sentence Comment

68 Portuguese CFTR alleles Spanish CFTR alleles Turkish CFTR alleles 5T 22 F508del 11 5T 20 F508del 14 5T 9 D1152H 14 R334W 5 D443Ya 3 D110H 3 R117H 3 G576Aa 3 F508del 2 S1235R 3 R668Ca 3 3041-11del7 2 N1303K 2 G542X 2 1767del6 2 P205S 2 R117H 2 2789þ5G>A 2 D614G 2 V232D 2 CFTRdele2(ins186) 2 G542X 1 L997F 1 3120þ1G>A 1 L206W 1 H609R 1 G1130A 1 V562I 1 N1303H 1 M952I 1 I507del 1 L206W 1 365insT 1 3272-26A>G 1 3272-26A/G 1 E585X 1 2789þ5G>A 1 L15P 1 2752-15C>G 1 G576Aa 1 R347H 1 R334Q 1 R668Ca 1 2689insG 1 R347H 1 CFTRdele2,3 1 R1070W 1 E831X 1 L1227S 1 I 1027T 1 R1070W 1 E831X 1 3272-26A>G 1 L997F 1 I853F 1 A349V 1 6T 1 Note: CFTR ¼ cystic fibrosis transmembrane conductance regulator. Login to comment

PMID: 20932301 [PubMed] Green DM et al: "Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients."

No. Sentence Comment

74 For Pa, the hazard ratio Table 1 Classification of CFTR alleles Category Mutation Specific mutations Class I Defective Protein Synthesis (nonsense, frameshift, aberrant splicing) 1078delT, 1154 insTC, 1525-2A > G, 1717-1G > A, 1898+1G > A, 2184delA, 2184 insA, 3007delG, 3120+1G > A, 3659delC, 3876delA, 3905insT, 394delTT, 4010del4, 4016insT, 4326delTC, 4374+1G > T, 441delA, 556delA, 621+1G > T, 621-1G > T, 711+1G > T, 875+1G > C, E1104X, E585X, E60X, E822X, G542X, G551D/R553X, Q493X, Q552X, Q814X, R1066C, R1162X, R553X, V520F, W1282X, Y1092X Class II Abnormal Processing and Trafficking A559T, D979A, ΔF508, ΔI507, G480C, G85E, N1303K, S549I, S549N, S549R Class III Defective Channel Regulation/Gating G1244E, G1349D, G551D, G551S, G85E, H199R, I1072T, I48T, L1077P, R560T, S1255P, S549 (R75Q) Class IV Decreased Channel Conductance A800G, D1152H, D1154G, D614G, delM1140, E822K, G314E, G576A, G622D, G85E, H620Q, I1139V, I1234V, L1335P, M1137V, P67L, R117C, R117P, R117H, R334W, R347H, R347P, R347P/ R347H, R792G, S1251N, V232D Class V Reduced Synthesis and/or Trafficking 2789+5G > A, 3120G > A, 3272-26A > G, 3849+10kbC > T, 5T variant, 621+3A > G, 711+3A > G, A445E, A455E, IVS8 poly T, P574H was increased 3 fold for those with 'Minimal` function when compared to those with 'Residual` function. Login to comment

PMID: 21642448 [PubMed] Caldwell RA et al: "Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators."

No. Sentence Comment

35 The folding defect caused by the V232D mutation appears to be due to the introduction of a charged residue into a region of CFTR that is embedded in the lipid bilayer of the ER membrane (17, 24). Login to comment
36 Since the folding defect in CFTR caused by the V232D mutation is correctable to wild-type levels, CF patients with this allele may benefit from treatment with folding correctors. Login to comment
154 Yet, the mechanism by which the V232D mutation causes CF is not well documented and whether patients with this mutation can be treated with modulators of CFTR folding and channel activity is not clear. Login to comment
156 The V232D mutation is proposed to cause aberrant hydrogen bonding between TM4 and adjacent TM segments in CFTR (24). Login to comment
165 Thus, CF patients that harbor low frequency mutations, such as V232D, might be treated with small molecules that correct CFTR misfolding or potentiate CFTR channel activity. Login to comment
169 In doing so, Corr-4a may prevent the V232D mutation from causing the aberrant hydrogen bonding between TM segments in the bilayer proposed to cause premature degradation of CFTRV232D (24). Login to comment
37 The folding defect caused by the V232D mutation appears to be due to the introduction of a charged residue into a region of CFTR that is embedded in the lipid bilayer of the endoplasmic reticulum (ER) membrane (17, 24). Login to comment
38 Since the folding defect in CFTR caused by the V232D mutation is correctable to wild-type levels, CF patients with this allele may benefit from treatment with folding correctors. Login to comment
191 Yet the mechanism by which the V232D mutation causes CF is not well documented, and whether patients with this mutation can be treated with modulators of CFTR folding and channel activity is not clear. Login to comment
193 The V232D mutation is proposed to cause aberrant hydrogen bonding between TM4 and adjacent TM segments in CFTR (24). Login to comment
202 Thus CF patients that harbor low-frequency mutations, such as V232D, might be treated with small molecules that correct CFTR misfolding or potentiate CFTR channel activity. Login to comment
206 In doing so, Corr-4a may prevent the V232D mutation from causing the aberrant hydrogen bonding between TM segments in the bilayer proposed to cause premature degradation of CFTRV232D (24). Login to comment

PMID: 9736775 [PubMed] Larriba S et al: "Testicular CFTR splice variants in patients with congenital absence of the vas deferens."

No. Sentence Comment

18 RESULTS CFTR analysis Eight different mutations (R117H, L206W, V232D, ∆F508, G542X, 711+1G→T, D1270N and 2789+5G→A) were found in nine of the 12 CBAVD patients, yielding a CFTR mutation frequencyof75%.ThreepatientspresentedtwoCFTRmutations, with one of them homozygous for the V232D mutation. Login to comment
26 CFTR genotype, IVS8-6 poly(T) allele and proportion of exon 9+ (E9+) and exon 9- (E9-) CFTR transcripts in testicular and epididymal biopsies Sample Phenotype CF mutation IVS8-6(T) Testis Epididymis n E9+ (%) E9- (%) n E9+ (%) E9- (%) 1 Non-CBAVD N/N 9T/9T 5 99 ± 0 1 ± 0 2 Non-CBAVD N/N 7T/7T 2 96 ± 2 4 ± 2 3 Non-CBAVD N/N 7T/7T 3 98 ± 0 2 ± 0 4 Non-CBAVD N/N 7T/7T 3 97 ± 1.5 3 ± 1.5 5 Non-CBAVD R334W/N 7T/7T 3 94 ± 1 6 ± 1 6 Non-CBAVD N/N 7T/7T 2 95 ± 1 5 ± 1 7 CBAVD V232D/V232D 9T/9T 4 96 ± 1.5 4 ± 1.5 8 CBAVD ∆F508/N 9T/9T 2 99 ± 0 1 ± 0 9 CBAVD ∆F508/D1270N 7T/9T 2 98 ± 1 2 ± 1 10 CBAVD G542X/2789+5G→A 7T/9T 2 96 ± 1 4 ± 1 11 CBAVD N/N 7T/7T 3 96 ± 2 4 ± 2 2 90 ± 3 10 ± 3 12 CBAVD N/N 7T/7T 2 94 ± 2 6 ± 2 5 78 ± 5 22 ± 5 13 CBAVD R117H/N 7T/7T 2 99 ± 0 1 ± 0 4 95 ± 2 5 ± 2 14 CBAVD G542X/5T 5T/9T 3 30 ± 2 70 ± 2 15 CBAVD ∆F508/5T 5T/9T 2 80 ± 5 20 ± 5 16 CBAVD L206W/5T 5T/9T 2 58 ± 2 42 ± 2 17 CBAVD 711+1G→T/5T 5T/7T 3 77 ± 4 23 ± 4 18 CBAVD 5T/N 5T/7T 5 71 ± 2 29 ± 2 The mean proportion of E9+ and E9- CFTR transcripts is calculated as the mean of the proportions found for each sample. Login to comment

PMID: 18596043 [PubMed] Loo TW et al: "Arginines in the first transmembrane segment promote maturation of a P-glycoprotein processing mutant by hydrogen bond interactions with tyrosines in transmembrane segment 11."

No. Sentence Comment

312 For example, it was demonstrated that the cystic fibrosis-phenotypic mutation V232D in CFTR TM segment 4 likely inhibits folding of CFTR by forming an unnatural hydrogen bond with Gln-207 in TM3 (47). Login to comment

PMID: 17331079 [PubMed] Alonso MJ et al: "Spectrum of mutations in the CFTR gene in cystic fibrosis patients of Spanish ancestry."

No. Sentence Comment

9 Furthermore, two other relatively common mutations (p.V232D and c.2789 + 5G > A) showed uneven geographic distributions. Login to comment
45 (%) p.F508del # E.10 1009 (51.74) p.G542X # E.11 150 (7.69) p.N1303K # E.21 57 (2.92) c.1811 + 1.6kbA > G I.11 36 (1.84) p.R334W # E.7 35 (1.79) p.L206W E.6a 32 (1.64) c.711 + 1G > T # I.5 31 (1.58) p.Q890X E.15 28 (1.43) p.R1162X # E.19 25 (1.28) c.2789 + 5G > A # I.14b 24 (1.23) p.R1066C E.17b 23 (1.18) p.I507del # E.10 21 (1.07) c.1609delCA E.10 18 (0.92) c.712-1G > T I.5 18 (0.92) c.3272-26A > G I.17a 18 (0.92) c.2183AA > G # E.13 16 (0.82) p.G85E # E.3 15 (0.77) c.2869insG E.15 15 (0.77) p.W1282X # E.20 15 (0.77) p.V232D E.6a 14 (0.71) p.A1006E * E.17a 12 (0.61) c.2184insA E.13 11 (0.56) p.K710X E.13 11 (0.56) TOTAL (n = 23) 1,634 (83.72) * , the complex allele [p.A1006E; p.V562I; IVS8-6(5T)] #, CF mutations identified with the Celera Diagnosis Cystic Fibrosis v2 genotyping assay and the Inno-Lipa CFTR12, CFTR17 + Tn Samples with microsatellite haplotypes 16/45-46-47 (IVS8CA/IVS17bTA) were submitted to direct analysis of the c.1811 + 1.6kbA > G mutation, which was found mainly associated with the 16-46 haplotype. Login to comment
62 Two mutations, p.V232D and c.1341G > A, showed the same high frequency (3/122, 2.5%) in the Castilla-Leon region. Login to comment
85 In addition, three other mild mutations, c.3272-26A > G, p.V232D and p.A1006E, showed frequencies ranging from 0.9% to 0.6% (Table 1). Login to comment
93 In addition, two other relatively frequent mutations showed unequal geographic distributions, p.V232D in the Castilla-Leon region (2.5%) and c.2789 + 5G > A in the Balearic Islands (10.5%). Login to comment

PMID: 22138447 [PubMed] Loo TW et al: "Corrector-mediated rescue of misprocessed CFTR mutants can be reduced by the P-glycoprotein drug pump."

No. Sentence Comment

14 Here, we tested our predictions that (1) other CFTR mutants such V232D and H1085R were more stable at the cell surface than DF508 CFTR after low temperature rescue and (2) the advantages of rescue with specific correctors (pharmacological chaperones) are that they may stabilize DF508 CFTR and increase the effectiveness of the correctors by bypassing drug pumps such as P-glycoprotein (P-gp) (increased bioavailability). Login to comment
15 It was found that the stability of mutants V232D and H1085R after low-temperature (30 8C) rescue was about 10-fold higher than DF508 CFTR. Login to comment
37 We show that rescue of DF508 CFTR with specific correctors resulted in a more stable protein and mutations such as V232D and H1085R are different from DF508 because they do not destabilize the rescued form of mature CFTR. Login to comment
106 (B) Samples of cells expressing DF508, V232D or H1085R CFTR mutants or P-gp mutant G268V and grown in the absence (À) or presence (+) of 20 mM VX-809 for 18 h were subjected to immunoblot analysis. Login to comment
120 We also tested whether correctors would promote maturation of CFTR mutants DF508, V232D and H1085R in HEK 293 cells in parallel because it has been reported that CFTR rescue depends on the cell system used [38-41]. Login to comment
121 Mutants V232D (TMD1) and H1085R (TMD2) were included because they are processing mutations located in different domains of CFTR (Fig. 1A) and both yield active proteins after rescue [16,42,43]. Login to comment
181 Degradation of wild-type, V232D, and H1085R CFTRs was monitored over time at 37 8C. Login to comment
203 To test if other CF mutations affect stability of CFTR, mutants V232D (TMD1) and H1085R (TMD2) were selected for study as both are processing mutations that yield active proteins after rescue [16,43]. Login to comment
204 Accordingly, we examined the stability of mutants V232D and H1085R after low-temperature rescue. Login to comment
205 Cells expressing wild-type, V232D, or H1085R CFTRs were expressed at low temperature to promote maturation of the protein. Protein synthesis was stopped by addition of cycloheximide, and turnover of the protein was monitored after incubation for 0-32 h at 37 8C (Fig. 7). Login to comment
206 It was observed that the half-lives of the mature forms of V232D and H1085R were at least 10-fold longer (about 14 and 12 h, respectively) than DF508 CFTR (about 1 h, Fig. 4). Login to comment
208 The V232D TMD1 and H1085R mutations may have less effect on the stability of mature CFTR because they have more localized effects on protein folding in the TMD1 and TMD2 domains, respectively. Login to comment
245 Corr-4a also may promote interactions between TMD1 and TMD2 as it restored folding of a processing mutant containing a charged residue (V232D) within TM4 [43] that is embedded in the lipid bilayer [56]. Login to comment
246 The V232D mutation is predicted to alter packing of the TM segments [56]. Login to comment

PMID: 22658665 [PubMed] Ooi CY et al: "Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in pancreatitis."

No. Sentence Comment

855 CFTR mutation Total PI Total PI + PS PIP score CFTR mutation Total PI Total PI + PS PIP score 621+1G>T 96 96 1.00 G542X 74 75 0.99 711+1G>T 36 36 1.00 F508del 1276 1324 0.96 I507del 34 34 1.00 1717-1G>A 20 21 0.95 R553X 24 24 1.00 W1282X 19 20 0.95 Q493X 11 11 1.00 N1303K 45 48 0.94 S489X 11 11 1.00 R1162X 12 13 0.92 1154insTC 10 10 1.00 Y1092X 12 13 0.92 3659delC 9 9 1.00 I148T 10 11 0.91 CFTRdele2 7 7 1.00 V520F 9 10 0.90 4016insT 7 7 1.00 G551D 59 67 0.88 E60X 7 7 1.00 L1077P 5 6 0.83 R560T 7 7 1.00 R1066C 5 6 0.83 R1158X 7 7 1.00 2184insA 9 12 0.75 3905insT 6 6 1.00 2143delT 3 4 0.75 I148T;3199del6 5 5 1.00 1161delC 3 4 0.75 2183AA>G 5 5 1.00 3120+1G>A 3 4 0.75 1898+1G>A 5 5 1.00 S549N 3 4 0.75 2347delG 4 4 1.00 G85E 16 22 0.73 Q1313X 3 3 1.00 R117C 2 3 0.67 Q220X 3 3 1.00 M1101K 19 30 0.63 2184delA 3 3 1.00 P574H 3 5 0.60 1078delT 3 3 1.00 474del13BP 1 2 0.50 L1254X 3 3 1.00 R352Q 1 2 0.50 E585X 3 3 1.00 Q1291H 1 2 0.50 3876delA 2 2 1.00 A455E 18 37 0.49 S4X 2 2 1.00 R347P 6 15 0.40 R1070Q 2 2 1.00 2789+5G>A 6 16 0.38 F508C 2 2 1.00 L206W 6 18 0.33 DELI507 2 2 1.00 IVS8-5T 4 16 0.25 Q1411X 2 2 1.00 3272-26A>G 1 4 0.25 365-366insT 2 2 1.00 R334W 1 10 0.10 R709X 2 2 1.00 3849+10kbC>T 2 22 0.09 1138insG 2 2 1.00 P67L 1 14 0.07 CFTRdele2-4 2 2 1.00 R117H 1 25 0.04 3007delG 2 2 1.00 R347H 0 5 0.00 Q814X 2 2 1.00 G178R 0 3 0.00 394delTT 2 2 1.00 E116K 0 2 0.00 406-1G>A 2 2 1.00 875+1G>C 0 2 0.00 R75X 2 2 1.00 V232D 0 2 0.00 CFTRdel2-3 2 2 1.00 D579G 0 2 0.00 E193X 2 2 1.00 L1335P 0 2 0.00 185+1G>T 2 2 1.00 Mild mutations (based on PIP scores) are shaded in gray. Login to comment

PMID: 22779403 [PubMed] Nadeau VG et al: "Sequence hydropathy dominates membrane protein response to detergent solubilization."

No. Sentence Comment

87 The intensity of mean residue ellipticity at 222 nm, for example, ranged by ~20 000 deg cm2 dmol-1 among the hairpin sequences (compare E217V and V232D, Figure 2A, left), a result that might not initially be anticipated for a "mild" detergent. Login to comment
89 We further noted that the spectra of mutant hairpins solubilized in DDM exhibited a gradation of intensities, whereas SDS-solubilized mutant spectra could be divided into "low helicity" (WT, V232D, A204L, and P205S) or "high helicity" (E217V, ES/SE, and E217F) clusters. Login to comment
107 The WT and mutant sequences complexed with DDM exhibited variable elution volumes, with ES/SE and V232D eluting earliest and latest from the column, respectively (Figure 3B, left). Login to comment
119 However, we noted that the elution volumes of the V232D mutant (open circle, Figure 3B) placed this hairpin outside the 95% confidence interval of the line of best fit. Login to comment
120 TM3/4-V232D can therefore be considered to differ in its hydrodynamic properties following solubilization by DDM vs SDS. Login to comment
165 N = 7 for the plots; the line of best fit omits the outlier mutant V232D (open circle, data point outside the 95% confidence interval of the fit). Login to comment
167 Note that the line of best fit has a near- unity slope, indicating that the relative order of elution is the same among the hairpin-DDM and hairpin-SDS complexes when V232D is excepted. Login to comment
172 Hydropathy Dependence in Local Sequence Context: The Cases of ES/SE and V232D. Login to comment
173 The ES/SE-DDM and V232D mutants are distinguished among the groups of DDM- or SDS-solubilized hairpins in that each of these hairpin-detergent complexes exhibits an apparent excluded volume on SEC that is larger than expected based on their rankings in helicity and low E:M ratios (compare Figures 3B, 2B, and 4B). Login to comment
174 Because the excluded volume of protein-detergent complexes is not an absolute measure of binding stoichiometry or particle size when particles deviate from compact, spherical shapes, we can only conclude at present that the shapes and/or dynamics of the ES/SE-DDM complex and the V232D-SDS complex differ in some way from the hairpin-DDM and hairpin-SDS complexes of equivalent helicities. Login to comment

PMID: 21909392 [PubMed] Roth EK et al: "The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients."

No. Sentence Comment

46 CFabsent CFresidual CFTR genotype Number of individuals CFTR genotype Number of individuals F508del/F508del 10 F508del/Y161C 1 F508del/W57X 1 F508del/V232D 1 F508del/G85E 3 F508del/R334W 2 F508del/120del23 1 F508del/T338I 1 F508del/182delT 1 F508del/I1234V 1 F508del/G542X 1 F508del/3272-26 A.G 1 F508del/A561E 1 F508del/3849+10 kb C.T 1 F508del/Y1092X 1 F508del/4005 +5727 A.G 1 F508del/N1303K 1 F508del/G576A 1 F508del/1525-1 G.A 2 N1303K/R334W 1 F508del/Q39X 1 F1052V/M1137R 1 F508del/Q552X 1 1898+3 A.G/ 1898+3 A.G 1 G85E/G85E 1 R334W/3199del6 1 Q552X/R1162X 1 R334W/X 1 A561E/A561E 2 dele2,3/X 1 R764X/1717-1 G.A 1 R1158X/2183AA.G 1 R1158X/R560T 1 doi:10.1371/journal.pone.0024445.t001 luminal and basolateral surfaces of the epithelium were perfused continuously with a solution of the following composition (mmol/ L): NaCl 145, KH2PO4 0.4, K2HPO4 1.6, D-glucose 5, MgCl2 1, Ca-gluconate 1.3, pH 7.4, at 37uC. Login to comment

PMID: 19733108 [PubMed] Grant CV et al: "A Modified Alderman-Grant Coil makes possible an efficient cross-coil probe for high field solid-state NMR of lossy biological samples."

No. Sentence Comment

94 The initial samples containing the TM3/4 V232D segment of CFTR would form ordered and well-aligned bicelles only at low concentrations, near the threshold of solid-state NMR detection. Login to comment
111 (A) One-dimensional 15 N chemical shift spectrum of uniformly 15 N-labeled CFTR TM3/4 V232D. Login to comment
124 Expression and purification The 15 N labeled form of the CFTR TM3/4 V232D helical hairpin construct was expressed and purified as previously described [26-28]. Login to comment
130 Prior to cell growth, the medium was supplemented with biotin and thiamine (1 mg/L of each); sterile MgSO4 and CaCl2 stock solutions to final concentrations of 1 mM and 0.3 mM, respectively; 3 g of glucose for expression of 15 N isotopically labeled TM3/4 V232D. Login to comment
136 Thrombin-treated TM3/4 V232D was purified by RP-HPLC on a C4 semipreparative column (Phenomenex) using an acetonitrile gradient. Login to comment
140 18 mg of TM3/4 V232D (>95% pure) per 1 L of minimal M9 medium. Login to comment
144 Bicelle sample preparation The uniformly 15 N-labeled TM3/4 V232D segment of CFTR and Pf1 major coat protein were reconstituted into bicelles as previously described [31,32]. Login to comment

PMID: 18456578 [PubMed] Castellani C et al: "Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice."

No. Sentence Comment

1236 Table 1 Geographical distribution of the most common mutations E60X Southern European S549N Indian CFTR Slavic - Eastern European G551D United Kingdom, Central Europe R75X Southern European, US-Hispanic Q552X Southern European, Italian 394delTT Nordic - Baltic sea region R553X Central European G85E Southern Europe A559T African-American 406-1GNA US-Hispanic R560T Northern Irish R117H European-derived populations 1811+1.6kbANG Spanish, US-Hispanic R117C Northern European 1898+1GNA United Kingdom, Central Europe 621+1GNT Southern European 1898+5GNT East Asian populations 711+1GNT French, French Canadian 2143delT Slavic - Eastern European 711+5GNA US-Hispanic 2183delAANG Southern Europe, Middle Eastern, Iranian, Latin American L206W Spanish and US-Hispanic 2184delA European-derived populations V232D Spanish and US-Hispanic 2789+5GNA European-derived populations 1078delT French Brittany Q890X Southern European R334W Southern European, Latin American 3120+1GNA African, Arabian, African-American, Southern Europe 1161delC Indian 3272-26ANG European-derived populations R347P European-derived, Latin America 3659delC Scandinavian R347H Turkish 3849+10kbCNT Ashkenazi-Jewish, Southern European, Middle Eastern, Iranian, Indian A455E Dutch R1066C Southern European 1609delCA Spanish, US-Hispanic Y1092X (CNA) Southern European I506T Southern European, Spanish M1101K US-Hutterite I507del European-derived populations 3905insT Swiss F508del European-derived populations D1152H European-derived populations 1677delTA Southern European, Middle Eastern R1158X Southern European 1717-GNA European-derived populations R1162X Italian, Amerindian, Latin America V520F Irish S1251N European-derived populations G542X Southern European, Mediterranean W1282X Ashkenazi-Jewish, Middle Eastern S549R(TNG) Middle Eastern N1303K Southern European, Middle Eastern Legend: these alleles occur with a frequency superior to 0.1% in selected populations. Login to comment
1239 Table 1 Geographical distribution of the most common mutations E60X Southern European S549N Indian CFTR Slavic - Eastern European G551D United Kingdom, Central Europe R75X Southern European, US-Hispanic Q552X Southern European, Italian 394delTT Nordic - Baltic sea region R553X Central European G85E Southern Europe A559T African-American 406-1GNA US-Hispanic R560T Northern Irish R117H European-derived populations 1811+1.6kbANG Spanish, US-Hispanic R117C Northern European 1898+1GNA United Kingdom, Central Europe 621+1GNT Southern European 1898+5GNT East Asian populations 711+1GNT French, French Canadian 2143delT Slavic - Eastern European 711+5GNA US-Hispanic 2183delAANG Southern Europe, Middle Eastern, Iranian, Latin American L206W Spanish and US-Hispanic 2184delA European-derived populations V232D Spanish and US-Hispanic 2789+5GNA European-derived populations 1078delT French Brittany Q890X Southern European R334W Southern European, Latin American 3120+1GNA African, Arabian, African-American, Southern Europe 1161delC Indian 3272-26ANG European-derived populations R347P European-derived, Latin America 3659delC Scandinavian R347H Turkish 3849+10kbCNT Ashkenazi-Jewish, Southern European, Middle Eastern, Iranian, Indian A455E Dutch R1066C Southern European 1609delCA Spanish, US-Hispanic Y1092X (CNA) Southern European I506T Southern European, Spanish M1101K US-Hutterite I507del European-derived populations 3905insT Swiss F508del European-derived populations D1152H European-derived populations 1677delTA Southern European, Middle Eastern R1158X Southern European 1717-GNA European-derived populations R1162X Italian, Amerindian, Latin America V520F Irish S1251N European-derived populations G542X Southern European, Mediterranean W1282X Ashkenazi-Jewish, Middle Eastern S549R(TNG) Middle Eastern N1303K Southern European, Middle Eastern Legend: these alleles occur with a frequency superior to 0.1% in selected populations. Login to comment

PMID: 17949679 [PubMed] Wehbi H et al: "Positional dependence of non-native polar mutations on folding of CFTR helical hairpins."

No. Sentence Comment

3 In the present work, we combine gel shift assays with a series of NMR experiments for comparative structural characterization of the wild type TM3/4 hairpin and its mutants V232D, I231D, Q207N/V232E. Login to comment
4 Over 95% of the backbone resonances of a 15 N,13 C-labelled V232D-TM3/4 construct in the membrane-mimetic environment of perfluorooctanoate (PFO) micelles were successfully assigned, and the presence and boundaries of helical segments within TM3 and TM4 were defined under these conditions. Login to comment
5 Comparative analysis of 15 N and 1 H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). Login to comment
13 CF is inherited in a recessive autosomal fashion; most CF patients have a CFTR Available online at www.sciencedirect.com Biochimica et Biophysica Acta 1778 (2008) 79-87 www.elsevier.com/locate/bbamem Abbreviations: CF, Cystic fibrosis; CFTR, Cystic fibrosis transmembrane conductance regulator; TM, Transmembrane; TMD, Transmembrane domain; TM3/4, Helical hairpin including residues 194-241 of CFTR; WT, Wild type; V232D-TM3/4, TM3/4 construct with mutation of Val to Asp at position 232; I231D-TM3/4, TM3/4 construct with mutation of Ile to Asp at position 231; Q207N/V232E-TM3/4, TM3/4 construct with mutation of Gln to Asn at position 207 and Val to Glu at position 232; PFO, Perfluorooctanoate; DPC, Dodecylphosphocholine; SDS, Sodium dodecylsulfate; CD, Circular dichroism; H-bond, Hydrogen bond; HSQC, Heteronuclear single quantum coherence ⁎ Corresponding author. Login to comment
35 Previous studies we performed on CFTR helix-loop-helix constructs using gel shift analysis, fluorescence measurements, and molecular modeling suggested that hairpin folding is likely stabilized by folding due to formation of a non-native side chain-side chain hydrogen bond between 'polar partners` in the interacting helices (viz., Q207 in TM3, and I231D or V232D in TM4) [20]. Login to comment
43 We then use solution NMR experiments to demonstrate the conformational variability of TM3/4 hairpin mutants (including the CF-phenotypic mutant V232D; I231D; and Q207N/V232E) vs. the wild type hairpin in micellar environments. Login to comment
46 Expression and purification of wild type and mutant TM3/4 constructs from the CFTR membrane domain The 15 N and 15 N/13 C enriched forms of the TM3/4 helical hairpin constructs (WT-, V232D-, I231D-, Q207N/V232E) were expressed and purified as previously described [19]. Login to comment
52 Prior to cell growth, the medium was supplemented with biotin and thiamine (1 mg/L of each); sterile MgSO4 and CaCl2 stock solutions to final concentrations of 1 mM and 0.3 mM, respectively; 3 g of glucose for expression of 15 N isotopically labeled V232D-TM3/4, or 13 C glucose (purchased from Cambridge Isotope Laboratories) for expression of 15 N/13 C isotopically labeled TM3/4; and 100 μg/mL ampicillin. Login to comment
73 Triple resonance NMR experiments for 15 N/13 C isotopically enriched V232D-TM3/4 115 mM PFO were carried out at 45 °C. Login to comment
94 Here, the double mutant Q207N/V232E migrates significantly faster than wild type, indicating that the N/E pair is likely involved in side chain-side chain interactions analogously to the Q/D pair at the corresponding positions in the TM3/4-V232D single mutant [22]. Login to comment
95 In contrast, replacement of wild type Q237 in TM4 with the non-polar Leu residue (mutant Q237L) creates a slower migrating hairpin vs. wild type (Fig. 1a), suggesting that some pre-existing inter-helical interactions - conceivably attributable to a network of H-bonding interactions - have been removed. Login to comment
99 Chemical shift assignment and secondary structure analysis of V232D-TM3/4 in PFO 1 H-15 N HSQC spectrum of 15 N-enriched V232D-TM3/4 in 115 mM PFO at 45 °C is shown in Fig. 2. Login to comment
104 Analysis of the triple resonance experiments HNCACB, CBCA(CO)NH, HNCO, (H)CC(CO)NH-TOCSY, and H(CC) (CO)NH-TOCSY of the 15 N,13 C-labeled V232D-TM3/4 enabled the assignments for 95% of the residues in the TM3/4 region, despite the fact that the analysis was complicated due to crosspeak overlapping typical of native helical TM proteins in detergent micelles. Login to comment
107 Hα, Cα, Cβ, CO, and NH chemical shifts of V232D-TM3/4 residues were analyzed using TALOS [51]. Login to comment
108 The secondary structure elements of V232D-TM3/4 thus obtained agree broadly with the formation of two helices in the micelle environment, separated by a turn. Login to comment
118 1 H-15 N HSQC spectrum of 15 N-labeled TM3/4 mutant V232D with amide chemical shift assignments. Login to comment
129 However, these NOEs were not sufficient per se for calculation of a defined tertiary structure for the V232D-TM3/4 hairpin. Login to comment
131 Analysis of 1 H-15 N HSQC spectra of WT-TM3/4 and its various mutants in PFO micelles To probe for global conformational changes of TM3/4 upon introducing a polar residue in TM4, the 1 H-15 N HSQC spectrum of wild type CFTR TM3/4 was acquired in PFO micelles under the same buffer conditions as V232D-TM3/4. Login to comment
132 With the amide chemical shift assignments of V232D-TM3/4 in hand, and because these two constructs differ by only a single residue, any major differences in the 1 H-15 N HSQC spectra should be attributable to the modification of the electrostatic environment by the non-polar-to-polar residue mutation at position 232, as well as to any resulting conformational changes. Login to comment
134 Summary of the chemical shift deviation and the sequential, mid-range NOEs for V232D-TM3/4 in PFO micelles. Login to comment
141 (a) WT; (b) V232D; (c) I231D; and (d) Q207N/V232E. Login to comment
142 Spectra were recorded in 115 mM PFO at 45 °C. Gly cross-peaks (shown in (b)) were assigned from the V232D spectrum (Fig. 2); other assignments shown were made where possible by comparison. Login to comment
144 As illustrated in the 'Gly box`, 1 H-15 N HSQC spectra of WT-TM3/ 4 (Fig. 4a) and V232D-TM3/4 (Fig. 4b) do possess some similarities. Login to comment
146 However, among the four spectra shown, significant chemical shift changes are observed for several of G213, G226, G228, G239 and G241 resonances between WT and V232D-TM3/4. Login to comment
147 As assignments were available only for the V232D-TM3/4 construct, we could not assign the full complement of WT Gly resonances specifically; however, the observed shifts are suggestive of a change of the environment of these residues. Login to comment
149 In order to further investigate the conformational changes of TM3/4 upon introduction of a polar residue into TM4, we next performed a comparative analysis of amide 1 H/15 N chemical shifts in the 1 H-15 N HSQC spectra of WT-TM3/4 vs. I231D-TM3/4 which contains a polar mutation at the position preceding the CF-phenotypic mutation V232D-TM3/4. Login to comment
150 Note that this mutant exhibited a faster migration rate than both WT-TM3/4 and V232D-TM3/4 on SDS-PAGE [22] (Fig. 1b). Login to comment
151 From the analysis of the Gly cross-peaks (Fig. 4c), one observes that the N-H chemical shifts of G3, G23, and G194 remain identical to the WT, but obvious changes occurred for G213, G226, G228, G239 and G241 to the extent that we could not formally assign them by comparison To V232D-TM3/4. Login to comment
153 We then examined the double mutant Q207N/V232E-TM3/4 in which the potential polar partners are located in the sequence at the same positions as in V232D-TM3/4 (Q207 in TM3 and D232 in TM4). Login to comment
154 This mutant similarly migrates faster than V232D-TM3/4 (Fig. 1a, b). Login to comment
155 As shown in Fig. 4b and d, a striking similarity was observed between the 1 H-15 N HSQC 'Gly box` spectra of V232D- and Q207N/V232E-TM3/4, suggesting that the cross-peaks of their Gly residues can be correspondingly assigned, and that their global conformations likely correspond closely. Login to comment
160 While some mutants do contain a change in charge vs. WT, previous work from our laboratory has established that CFTR TM3/4 migration patterns are not simple functions of charge: (i) WT TM3/4 and the double mutant Q207L/V232D the same migration rates while V232D migrates significantly faster than WT [20]; (ii) Asp substitutions at 20 different positions along TM4 between residues 221 and 241 produce TM3/4 hairpins that migrate 3-12% faster than WT [22]; if introduction of a single negative charge was the dominating effect, all 20 mutants should display similar migration rates. Login to comment
161 In the present work, Q207N/V232E-TM3/4 migrates faster than TM3/4-V232D (Fig. 1b) although they each have one added negative charge vs. WT. Login to comment
174 We further observed that the turn (approximately S222 to G226) determined between TM3 and TM4 in the V232D-TM3/4 construct is shifted approximately six residues toward TM4 vs. the one predicted (W216 to Q220) in the original schematic presented for the wild type CFTR protein [6]. Login to comment
187 Similarly, G194 present at the N-terminal end of TM3, is not affected by the V232D mutation (Fig. 4b). Login to comment
189 In addition to these "Gly box" effects, chemical shift changes vs. WT in the amide proton dimension of virtually all of the residues in the V232D-TM3/4 spectrum, including A223, A225, and G226 from the turn, as well as W216, L218, L219, Q220, and A221 near the C-terminus of the TM3 helix (data not shown), confirming that the effect is not limited to immediate neighbors. Login to comment
190 These changes, in concert with results from migration rate data on SDS-PAGE, emphasize the fact that the interactions between helices differ in WT- vs. V232D-TM3/4. Login to comment
192 To address the proposition that the formation of a given helix-helix interface may be dominated by a positional dependence of a polar residue in TM4, the 1 H-15 N HSQC spectrum of I231D-TM3/4 in PFO micelles was acquired under identical conditions as WT-TM3/4 and V232D-TM3/4. Login to comment
194 The full amide chemical shift analysis of the 1 H-15 N HSQC shows that similarly to the V232D mutant, all TM4 amides - along with residues from W216 through A221 (C-terminus of TM3) and from S222 to G226 (turn) - are highly affected by the I231D mutation (not shown), suggesting that this mutation introduces significant changes in packing relative to WT protein. Login to comment
195 In contrast, the full 1 H-15 N HSQC spectra for V232D- and Q207N/V232E-TM3/4 exhibit striking similarities (not shown, but exemplified by the 'Gly box` comparison between Fig. 4 b and d), suggesting that these two mutants adopt similar conformations in the PFO micelle environment. Login to comment
196 The 1 H-15 N HSQC spectrum of this double mutant shows that similar to V232D-TM3/4 and I231D-TM3/4 mutants, the residues in TM4 along with some in TM3 and the turn are all highly affected vs. WT upon these mutations. Login to comment
200 Although the NMR data reported here do not provide specific evidence for H-bond formation nor are sufficient for detailed structural characterization, one can speculate that the similarities in spectra of V232D and Q207N/V232E are the result of contacts between similar residues in the V232D mutant that can effectively be substituted by N207 and E232. Login to comment
202 Conversely, this model would predict that in the case of I231D, there must be a relative reorientation by 100° of one helix with respect to the other for participation of I231D in interhelical interactions vs. either V232D or Q207N/V232E. Login to comment
208 Conclusion Features of the secondary structure of mutant V232D-TM3/4 helical hairpins of CFTR, along with comparisons to the WT and mutants I2231D and Q207N/V232E, have been determined using high resolution NMR spectroscopy. Login to comment
209 Although hairpin constructs have degrees of conformational freedom in SDS or PFO micellar environments that would not be available to the corresponding helices embedded in an intact CFTR TM domain, our results suggest that hairpin interfaces - and possibly helix boundaries - may vary as a function of the position of a non-native polar mutation (i.e., V232D vs. I231D in TM4), and thereby indicate the susceptibility of the native protein structure to a corresponding destiny. Login to comment
6 Comparative analysis of 15 N and 1 H chemical shift variations among HSQC spectra of WT-, V232D-, I231D- and Q207N/V232E-TM3/4 indicated that hairpin conformations vary with the position of a polar mutation (i.e., V232D and I231D vs. WT), but remain similar when hairpins with identically-positioned polar partners are compared (i.e., V232D vs. Q207N-V232E). Login to comment
14 CF is inherited in a recessive autosomal fashion; most CF patients have a CFTR Available online at www.sciencedirect.com Biochimica et Biophysica Acta 1778 (2008) 79-87 www.elsevier.com/locate/bbamem Abbreviations: CF, Cystic fibrosis; CFTR, Cystic fibrosis transmembrane conductance regulator; TM, Transmembrane; TMD, Transmembrane domain; TM3/4, Helical hairpin including residues 194-241 of CFTR; WT, Wild type; V232D-TM3/4, TM3/4 construct with mutation of Val to Asp at position 232; I231D-TM3/4, TM3/4 construct with mutation of Ile to Asp at position 231; Q207N/V232E-TM3/4, TM3/4 construct with mutation of Gln to Asn at position 207 and Val to Glu at position 232; PFO, Perfluorooctanoate; DPC, Dodecylphosphocholine; SDS, Sodium dodecylsulfate; CD, Circular dichroism; H-bond, Hydrogen bond; HSQC, Heteronuclear single quantum coherence Ìe; Corresponding author. Login to comment
36 Previous studies we performed on CFTR helix-loop-helix constructs using gel shift analysis, fluorescence measurements, and molecular modeling suggested that hairpin folding is likely stabilized by folding due to formation of a non-native side chain-side chain hydrogen bond between 'polar partners` in the interacting helices (viz., Q207 in TM3, and I231D or V232D in TM4) [20]. Login to comment
44 We then use solution NMR experiments to demonstrate the conformational variability of TM3/4 hairpin mutants (including the CF-phenotypic mutant V232D; I231D; and Q207N/V232E) vs. the wild type hairpin in micellar environments. Login to comment
47 Expression and purification of wild type and mutant TM3/4 constructs from the CFTR membrane domain The 15 N and 15 N/13 C enriched forms of the TM3/4 helical hairpin constructs (WT-, V232D-, I231D-, Q207N/V232E) were expressed and purified as previously described [19]. Login to comment
53 Prior to cell growth, the medium was supplemented with biotin and thiamine (1 mg/L of each); sterile MgSO4 and CaCl2 stock solutions to final concentrations of 1 mM and 0.3 mM, respectively; 3 g of glucose for expression of 15 N isotopically labeled V232D-TM3/4, or 13 C glucose (purchased from Cambridge Isotope Laboratories) for expression of 15 N/13 C isotopically labeled TM3/4; and 100 bc;g/mL ampicillin. Login to comment
74 Triple resonance NMR experiments for 15 N/13 C isotopically enriched V232D-TM3/4 115 mM PFO were carried out at 45 &#b0;C. Login to comment
100 Chemical shift assignment and secondary structure analysis of V232D-TM3/4 in PFO 1 H-15 N HSQC spectrum of 15 N-enriched V232D-TM3/4 in 115 mM PFO at 45 &#b0;C is shown in Fig. 2. Login to comment
105 Analysis of the triple resonance experiments HNCACB, CBCA(CO)NH, HNCO, (H)CC(CO)NH-TOCSY, and H(CC) (CO)NH-TOCSY of the 15 N,13 C-labeled V232D-TM3/4 enabled the assignments for 95% of the residues in the TM3/4 region, despite the fact that the analysis was complicated due to crosspeak overlapping typical of native helical TM proteins in detergent micelles. Login to comment
109 The secondary structure elements of V232D-TM3/4 thus obtained agree broadly with the formation of two helices in the micelle environment, separated by a turn. Login to comment
119 1 H-15 N HSQC spectrum of 15 N-labeled TM3/4 mutant V232D with amide chemical shift assignments. Login to comment
130 However, these NOEs were not sufficient per se for calculation of a defined tertiary structure for the V232D-TM3/4 hairpin. Login to comment
133 With the amide chemical shift assignments of V232D-TM3/4 in hand, and because these two constructs differ by only a single residue, any major differences in the 1 H-15 N HSQC spectra should be attributable to the modification of the electrostatic environment by the non-polar-to-polar residue mutation at position 232, as well as to any resulting conformational changes. Login to comment
135 Summary of the chemical shift deviation and the sequential, mid-range NOEs for V232D-TM3/4 in PFO micelles. Login to comment
143 Spectra were recorded in 115 mM PFO at 45 &#b0;C. Gly cross-peaks (shown in (b)) were assigned from the V232D spectrum (Fig. 2); other assignments shown were made where possible by comparison. Login to comment
145 As illustrated in the 'Gly box`, 1 H-15 N HSQC spectra of WT-TM3/ 4 (Fig. 4a) and V232D-TM3/4 (Fig. 4b) do possess some similarities. Login to comment
148 As assignments were available only for the V232D-TM3/4 construct, we could not assign the full complement of WT Gly resonances specifically; however, the observed shifts are suggestive of a change of the environment of these residues. Login to comment
152 From the analysis of the Gly cross-peaks (Fig. 4c), one observes that the N-H chemical shifts of G3, G23, and G194 remain identical to the WT, but obvious changes occurred for G213, G226, G228, G239 and G241 to the extent that we could not formally assign them by comparison To V232D-TM3/4. Login to comment
156 As shown in Fig. 4b and d, a striking similarity was observed between the 1 H-15 N HSQC 'Gly box` spectra of V232D- and Q207N/V232E-TM3/4, suggesting that the cross-peaks of their Gly residues can be correspondingly assigned, and that their global conformations likely correspond closely. Login to comment
162 In the present work, Q207N/V232E-TM3/4 migrates faster than TM3/4-V232D (Fig. 1b) although they each have one added negative charge vs. WT. Login to comment
175 We further observed that the turn (approximately S222 to G226) determined between TM3 and TM4 in the V232D-TM3/4 construct is shifted approximately six residues toward TM4 vs. the one predicted (W216 to Q220) in the original schematic presented for the wild type CFTR protein [6]. Login to comment
188 Similarly, G194 present at the N-terminal end of TM3, is not affected by the V232D mutation (Fig. 4b). Login to comment
191 These changes, in concert with results from migration rate data on SDS-PAGE, emphasize the fact that the interactions between helices differ in WT- vs. V232D-TM3/4. Login to comment
193 To address the proposition that the formation of a given helix-helix interface may be dominated by a positional dependence of a polar residue in TM4, the 1 H-15 N HSQC spectrum of I231D-TM3/4 in PFO micelles was acquired under identical conditions as WT-TM3/4 and V232D-TM3/4. Login to comment
197 The 1 H-15 N HSQC spectrum of this double mutant shows that similar to V232D-TM3/4 and I231D-TM3/4 mutants, the residues in TM4 along with some in TM3 and the turn are all highly affected vs. WT upon these mutations. Login to comment
201 Although the NMR data reported here do not provide specific evidence for H-bond formation nor are sufficient for detailed structural characterization, one can speculate that the similarities in spectra of V232D and Q207N/V232E are the result of contacts between similar residues in the V232D mutant that can effectively be substituted by N207 and E232. Login to comment
203 Conversely, this model would predict that in the case of I231D, there must be a relative reorientation by 100&#b0; of one helix with respect to the other for participation of I231D in interhelical interactions vs. either V232D or Q207N/V232E. Login to comment
210 Although hairpin constructs have degrees of conformational freedom in SDS or PFO micellar environments that would not be available to the corresponding helices embedded in an intact CFTR TM domain, our results suggest that hairpin interfaces - and possibly helix boundaries - may vary as a function of the position of a non-native polar mutation (i.e., V232D vs. I231D in TM4), and thereby indicate the susceptibility of the native protein structure to a corresponding destiny. Login to comment

PMID: 17116404 [PubMed] Bond PJ et al: "Coarse-grained molecular dynamics simulations of membrane proteins and peptides."

No. Sentence Comment

220 It is thought that the Q207 sidechain in M3 may form an interhelical H-bond with a CF-phenotypic mutant V232D in M4 (Therien et al., 2001). Login to comment
219 It is thought that the Q207 sidechain in M3 may form an interhelical H-bond with a CF-phenotypic mutant V232D in M4 (Therien et al., 2001). Login to comment

PMID: 15858154 [PubMed] Schrijver I et al: "Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum."

No. Sentence Comment

83 While ⌬F508 was homozygous in six subjects, seven other less common alleles (G542X, W1204X, R75X, V232D, E116K, T501A, 3272-26 AϾG) were also seen in the homozygous state. Login to comment
103 Table 1. Continued Mutations in 257 patients Allele counts of each mutation % of variant alleles (183) % of all alleles tested (514) R1070W 1 0.55 0.19 R1158X 1 0.55 0.19 R1438W 1 0.55 0.19 R334W 2 1.09 0.39 R352W 1 0.55 0.19 R553X 2 1.09 0.39 R668C 2 1.09 0.39 R74W 3 1.64 0.58 R75X 3 1.64 0.58 S1235R 2 1.09 0.39 S492F 2 1.09 0.39 S549N 1 0.55 0.19 S573CS573C 1 0.55 0.19 S945L 1 0.55 0.19 T351S 1 0.55 0.19 T501A 2 1.09 0.39 T604ST604S 1 0.55 0.19 V11I 1 0.55 0.19 V201 mol/L 1 0.55 0.19 V232D 2 1.09 0.39 V754 mol/L 1 0.55 0.19 W1089X 2 1.09 0.39 W1098C 1 0.55 0.19 W1204X 4 2.19 0.78 Y563N 1 0.55 0.19 Y913XY913X 1 0.55 0.19 85 different mutations 183 100.00 35.60 Novel variants are in boldface, mutations on the ACMG/ACOG panel are italicized. Login to comment
187 CFTR Sequence Variants Identified in Five Comprehensive CFTR Studies in US Hispanics CFTR mutations Alleles Relative mutation frequency (%) (of 317) deltaF508 123 38.80 3876delA 15 4.70 G542X 12 3.80 406 - 1GϾA 8 2.50 3849 ϩ 10kbCϾT 5 1.60 R75X 4 1.30 935delA 4 1.30 S549N 4 1.30 W1204X 4 1.30 R334W 4 1.30 2055del9ϾA 3 1 R74W 3 1 H199Y 3 1 L206W 3 1 663delT 3 1 3120 ϩ 1GϾA 3 1 L997F 3 1 I1027T 3 1 R1066C 3 1 W1089X 3 1 D1270N 3 1 2105del13insAGAAA 3 1 Q98R 2 Ͻ1 E116K 2 Ͻ1 I148T 2 Ͻ1 R668C 2 Ͻ1 P205S 2 Ͻ1 V232D 2 Ͻ1 S492F 2 Ͻ1 T501A 2 Ͻ1 1949del84 2 Ͻ1 Q890X 2 Ͻ1 3271delGG 2 Ͻ1 3272 - 26AϾG 2 Ͻ1 G1244E 2 Ͻ1 D1445N 2 Ͻ1 R553X 2 Ͻ1 E588V 2 Ͻ1 1717 - 8GϾA 2 Ͻ1 A1009T 2 Ͻ1 S1235R 2 Ͻ1 G85E 1 Ͻ1 296 ϩ 28AϾG 1 Ͻ1 406 - 6TϾC 1 Ͻ1 V11I 1 Ͻ1 Q179K 1 Ͻ1 V201 mol/L 1 Ͻ1 874insTACA 1 Ͻ1 I285F 1 Ͻ1 deltaF311 1 Ͻ1 F311L 1 Ͻ1 L320V 1 Ͻ1 T351S 1 Ͻ1 R352W 1 Ͻ1 1248 ϩ 1GϾA 1 Ͻ1 1249 - 29delAT 1 Ͻ1 1288insTA 1 Ͻ1 1341 ϩ 80GϾA 1 Ͻ1 1429del7 1 Ͻ1 1525 - 42GϾA 1 Ͻ1 P439S 1 Ͻ1 1717 - 1GϾA 1 Ͻ1 1811 ϩ 1GϾA 1 Ͻ1 deltaI507 1 Ͻ1 G551D 1 Ͻ1 A559T 1 Ͻ1 Y563N 1 Ͻ1 (Table continues) In this study, we used temporal temperature gradient gel electrophoresis (TTGE) and direct DNA sequencing to increase the sensitivity of mutation detection in U.S. Hispanics, and to determine whether additional mutations are recurrent. Login to comment
201 Comparison of Relative Frequencies of CFTR Sequence Variants in Comprehensive CFTR Studies in US and Mexican Hispanics This study % Orozco 2000 % US/ Mexican % deltaF508 28.96 54.48 43.72 G542X 3.83 8.28 5.19 406 - 1GϾA 3.28 2.07 2.38 W1204X 2.19 Ͻ1 1.08 R74W 1.64 Ͻ1 R75X 1.64 2.07 1.51 H199Y 1.64 Ͻ1 Ͻ1 L206W 1.64 Ͻ1 L997F 1.64 Ͻ1 I1027T 1.64 Ͻ1 2055del9ϾA 1.64 1.38 1.27 D1270N 1.64 Ͻ1 E116K 1.09 Ͻ1 V232D 1.09 Ͻ1 R334W 1.09 Ͻ1 S492F 1.09 Ͻ1 T501A 1.09 Ͻ1 R553X 1.09 Ͻ1 Ͻ1 E588V 1.09 Ͻ1 R668C 1.09 Ͻ1 Q890X 1.09 Ͻ1 W1089X 1.09 Ͻ1 S1235R 1.09 Ͻ1 D1445N 1.09 Ͻ1 3876delA 1.09 3.24 1717 - 8GϾA 1.09 Ͻ1 3272 - 26AϾG 1.09 Ͻ1 A1009T 1.09 Ͻ1 deltaI507 Ͻ1 3.45 1.30 S549N Ͻ1 3.45 1.95 G567A Ͻ1 Ͻ1 I148T 2.07 1.08 I506T 1.38 Ͻ1 N1303K 2.76 1.08 935delA 1.38 1.30 2183AAϾG 1.38 Ͻ1 3199del6 1.38 Ͻ1 3849 ϩ 10kbCϾT Ͻ1 1.30 ACMG/ACOG italicized. Login to comment

PMID: 15480987 [PubMed] Hirtz S et al: "CFTR Cl- channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis."

No. Sentence Comment

78 Relationship Between the CFTR Genotype and Cl- Channel Function in Native Rectal Epithelia CFTR genotype Number of individuals Sweat Cl-concentration (mmol/L)a cAMP-mediated response Carbachol-induced plateau response or maximal lumen-negative response Isc-cAMP (␮A/cm2) Cl- secretion (% of control) Isc-carbachol (␮A/cm2) Cl- secretion (% of control) Cl- secretion absent R1162X/Q552X 1 71 17.1 0 0.7 0 W1282X/3121-2AϾG 1 112 1.9 0 0.6 0 1898 ϩ 1G Ͼ T/1609delCA 2b 114, 118 25.4, 13.4 0, 0 0, 0.7 0, 0 ⌬F508/Q39X 2b 127, 129 2.6, 4.4 0, 0 1.7, 3.7 0, 0 ⌬F508/G542X 1 102 29.0 0 6.6 0 ⌬F508/R553X 3 112, 102, 109 13.1, 4.5, 23.8 0, 0, 0 1.5, 4.4, 1.0 0, 0, 0 ⌬F508/E585X 1 115 1.4 0 1.1 0 ⌬F508/Q637X 1 100 2.9 0 1.2 0 ⌬F508/Y1092X 1 119 0.0 0 -0.3 0 ⌬F508/120del23c 1 72 20.1 0 3.3 0 ⌬F508/182delT 1 116 10.8 0 5.2 0 ⌬F508/3905insT 2 88, 96 8.4, 5.6 0, 0 2.3, -1.1 0, 1 ⌬F508/V520F 1 68 1.2 0 1.7 0 ⌬F508/A561E 3 113, 146, 100 17.0, 17.0, 16.0 0, 0, 0 2.1, 1.5, 3.7 0, 0, 0 ⌬F508/R1066C 1 138 0.0 0 0.0 0 ⌬F508/N1303K 3 100, 117, 94 1.7, 4.1, 1.5 0, 0, 0 -0.6, 2.2, 0.8 0, 0, 0 A561E/A561E 2 101, 116 6.6, 2.0 0, 0 7.3, 3.3 0, 0 Residual Cl- secretiond G542X/I148N 1 75 -50.1 54 -22.2 12 1898 ϩ 3A Ͼ G/1898 ϩ 3A Ͼ G 1 82 -36.8 39 -12.9 7 ⌬F508/3272-26A Ͼ G 1 116 -17.8 19 -27.2 14 ⌬F508/S108F 1 118 -15.8 17 -12.3 7 ⌬F508/R117H 1 90 -35.9 38 -207.7 109 ⌬F508/Y161Cc 1 44 -35.1 37 -45.9 25 ⌬F508/P205S 1 80 -23.3 25 -10.4 5 ⌬F508/V232D 1 120 -16.9 18 -26.9 14 ⌬F508/R334W 1 92 -22.1 23 -21.1 11 ⌬F508/R334W 1 101 -24.5 26 -37.4 20 ⌬F508/T338I 1 73 -44.4 47 -79.4 42 ⌬F508/G576A 1 40 -16.9 18 -115.5 61 ⌬F508/I1234V 1 113 -13.6 15 -8.6 5 G576A/G85E 1 95 -26.1 28 -61.6 32 F1052V/M1137R 1 47 -36.7 39 -146.6 77 M1101K/M1101K 1 94 -11.1 12 -4.8 3 S1159F/S1159F 1 67 -47.9 51 -38.7 21 N1303K/R334W 1 91 -30.3 32 -47.7 25 NOTE. CFTR Cl- channel function was determined in rectal epithelia from Cl- secretory responses induced by IBMX/forskolin (Isc-cAMP) and after co-activation with carbachol (Isc-carbachol). Login to comment
101 Functional Classification and Protein Location of CFTR Mutations Mutation type Severe mutations (protein location) Mild mutations (protein location) Missense V520F, A561E (NBD1) G85E (MSD1, TM1) R1066C (MSD2, CL4) S108F, R117H (MSD1, EL1) N1303K (NBD2) I148N, Y161Ca (MSD1, CL1) P205S (MSD1, TM3) V232D (MSD1, TM4) R334W, T338I (MSD1, TM6) G576A (NBD1) I1234V (NBD2) F1052V, M1101K (MSD2, CL4) M1137R (MSD2, TM12) S1159F (pre-NBD2) Splice 1898 ϩ 1G Ͼ T (R domain) 1898 ϩ 3A Ͼ G (R domain) 3121-2A Ͼ G (MSD2, TM9) 3272-26A Ͼ G (MSD2, TM10) Single amino acid deletion ⌬F508 (NBD1) Nonsense Q39X (N-terminus) G542X, Q552X, R553X, E585X (NBD1) Q637X (R domain) Y1092X (MSD2, CL4) R1162X (pre-NBD2) W1282X (NBD2) Frameshift 120del23a 182delT (N-terminus) 1609delCA (NBD1) 3905insT (NBD2) NOTE. Severe mutation, Cl- secretion absent; mild mutation, residual cAMP-mediated Cl- secretion. Login to comment
125 According to our functional data, 3121-2AϾG, 1898ϩ1GϾT, and V520F constitute severe mutations, whereas 1898ϩ3AϾG, I148N, Y161C, V232D, T338I, I1234V, and S1159F confer residual CFTR Cl- channel function (Table 1). Login to comment

PMID: 15209503 [PubMed] Choi MY et al: "Non-native interhelical hydrogen bonds in the cystic fibrosis transmembrane conductance regulator domain modulated by polar mutations."

No. Sentence Comment

2 Here we have undertaken an investigation of the presence and strength of H-bond interactions within a series of helix-loop-helix ("hairpin") constructs derived from TM helices 3 and 4 (italic) of the cystic fibrosis transmembrane conductance regulator (CFTR) (prototypic sequence G194LALAHFVWIAPLQ207VALLMGLIWELLQASAFAGLGFLIV232LALFQ237AGLG241) in which wild-type Q207 in TM3 forms an interhelical H-bond with CF-phenotypic mutant V232D in TM4 [Therien, A. G., Grant, F. E., and Deber, C. M. (2001) Nat. Struct. Biol 8, 597-601]. Login to comment
28 1 Abbreviations: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; TM, transmembrane; VD, V232D; wt, wild type; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; CD, circular dichroism; H-bond, hydrogen bond; RP-HPLC, reversed-phase high-performance liquid chromatography; LB, Luria-Bertani; MW, molecular weight; TFE, trifluoroethanol. Login to comment
33 With the use of this "gel-shift assay" on SDS-PAGE, we observed that the construct derived from the phenotypic mutant Val232-Aspswhich induces a mild form of CFsmigrated significantly faster than the wt construct (22), indicating that V232D adopts a more compact conformation likely imposed by a tight interaction between the mutant Asp 232 in TM helix 4 and its polar partner Gln 207 in TM helix 3 (22). Login to comment
81 Previous studies have shown that the V232D mutant migrates at a rate faster than that of the wt in NuPAGE MES gels (22). Login to comment
82 This migration pattern was shown to be associated with a more compact conformation of the V232D construct, imposed by a tight interaction between polar partners Asp 232 and Gln 207 of the V232D TM3/4 construct. Login to comment
89 Interestingly, mutants G228D through I231D, and L233D through F236D, have a more negative value of the percent of MW decrease relative to the wt than the CF-phenotypic mutant V232D (Figure 3b) (see the Discussion). Login to comment
90 The remaining Asp mutants toward either terminus of the TM4 helix migrated slower than the V232D construct on SDS-PAGE. Login to comment
97 V232D is the original CF-phenotypic mutant studied by Therien et al. (22). Login to comment
104 V232D is the CF-phenotypic mutant in TM4. Login to comment
111 Double asterisks denote values statistically different from that of the construct V232D on SDS-PAGE gel (p < 0.01). Login to comment
120 D mutants in mid-TM4, i.e., V232D/Q237L and L230D/Q237L, continued to migrate faster than the wt, but were clearly retarded vs their D counterparts. Login to comment
121 DISCUSSION In a helical hairpin construct derived from the first transmembrane domain of CFTR, the CF-phenotypic mutant V232D in TM4 was previously found to form a non-native side chain-side chain H-bond with the wild-type residue Q207 in TM3 (22) which was proposed to be responsible, in part, for the disruption of the functional CFTR protein. Login to comment
178 From the results (Figures 5 and 7), we found that double mutants where D is relatively distant from Q207 in TM3 (F224D/ Q237L, A238D/Q237L, and G239D/Q237L) returned to the wt migration rate, whereas the other double mutants (V232D/ Q237L and L230D/Q237L) migrated more slowly than the corresponding D mutants, but still significantly faster than the wt construct. Login to comment
182 The CF-phenotypic Mutant V232D Is Not the Optimal Position for Interhelix H-Bond Formation. Login to comment
183 As elaborated in Figure 3b, the CF-phenotypic mutant V232D does not form the tightest H-bond with Q207 in TM3 in comparison to the several X f D mutants on TM4. Login to comment
184 In fact, the migration rate of V232D is statistically slower vs those of D mutants at 229, 230, 231, and 233 (double asterisks, Figure 3b). Login to comment
186 As V232D is the CF-phenotypic mutant in this series, this result initially appeared counterintuitive. Login to comment
189 It is also interesting to note that several mutantssincluding S222D, G226D, V232D, and Q237Dsdisplay locally slower migration rates. Login to comment
191 One might speculate that this is the case for CF-phenotypic mutant V232D, and therefore, its capture by Q207 necessitates major reorientation of the wild-type TM4 axissand accordingly the greatest disruption of the native folded domain structure. Login to comment

PMID: 12071702 [PubMed] Therien AG et al: "Expression and purification of two hydrophobic double-spanning membrane proteins derived from the cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

91 These constructs have proven valuable in studies of the secondary and tertiary structure of wild-type and mutant CFTR, as in our recent investigation of the structural consequences of the cystic fibrosis-phenotypic mutant V232D in TM 3-4 (9). Login to comment

PMID: 10923036 [PubMed] Claustres M et al: "Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France."

No. Sentence Comment

108 g D44G, 300delA, W57X, 405+1G>A, D110H, E116K, 541del4, 542del7, L137R, 621+2T>G, I175V, H199R, H199Y, C225X, V232D, Q290X, E292X, G314V, T338I, 1221delCT, W401X, Q452P, I502T, 1716+2T>C, G544S, R560S, A561E, V562I, Y569D, 1898+3A>G, 1898+5G>A, G628R(G>A), 2143delT, G673X, R851X, Q890X, S977F, 3129del4, 3154delG, 3271+1G>A, G1061R, R1066L, R1070W, 3601-17T>C, S1196X, 3732delA, G1249R, 3898insC, 4374+1G>A, del25kb. Login to comment
152 Twenty-four non F508del mutations were found associated with the 9T allele: 394delTT, L90S, D110H, R117G, 621+1G>T, V232D, A455E, G542X, R851L, T908N, 2789+5G>A, 2896insAG, H939R, 3007delG, I980K, I1027T, R1066H, A1067T, D1154G, 3737delA, R74W+D1270N, N1303I, N1303K, D1377H. Login to comment

PMID: 9439669 [PubMed] Casals T et al: "High heterogeneity for cystic fibrosis in Spanish families: 75 mutations account for 90% of chromosomes."

No. Sentence Comment

33 Eight mutations have frequencies 366 Table 1 Seventy-five CFTR mutations identified in 640 Spanish families with cystic fibrosis (CF) Mutation Exon/intron CF alleles % ∆F508 E.10 681 53.20 G542X E.11 108 8.43 N1303K E.21 34 2.65 1811+1.6kbA→Ga I.11 24 1.87 711+1G→T I.5 22 1.71 R1162Xa E.19 21 1.64 R334Wa E.7 21 1.64 R1066C E.17b 14 1.09 1609delCAa E.10 13 1.01 Q890X E.15 13 1.01 G85E E.3 12 0.94 712-1G→Ta I.5 11 0.86 2789+5G→A I.14b 11 0.86 ∆I507 E.10 10 0.78 W1282X E.20 10 0.78 2869insGa E.15 9 0.70 L206W E.6a 7 0.54 R709X E.13 7 0.54 621+1G→T I.4 6 0.47 3272-26A→G I.17a 6 0.47 R347H E.7 5 0.39 2183AA→G E.13 5 0.39 K710X E.13 5 0.39 2176insC E.13 5 0.39 3849+10kbC→T I.19 5 0.39 P205Sa E.6a 4 0.31 1078delT E.7 4 0.31 R553X E.11 4 0.31 G551D E.11 4 0.31 1812-1G→Aa I.11 4 0.31 CFdel#1a E.4-7/11-18 4 0.31 V232D E.6a 3 0.23 936delTAa E.6b 3 0.23 1717-8G→A I.10 3 0.23 1949del84 E.13 3 0.23 W1089X E.17b 3 0.23 R347P E.7 3 0.23 del E.3a E.3 2 0.16 R117H E.4 2 0.16 L558S E.11 2 0.16 A561E E.12 2 0.16 2603delT E.13 2 0.16 Y1092X E.17b 2 0.16 Q1100Pa E.17b 2 0.16 M1101K E.17b 2 0.16 delE.19a E.19 2 0.16 G1244E E.20 2 0.16 P5La E.1 1 0.08 Q30Xa E.2 1 0.08 G85Va E.3 1 0.08 E92Ka E.4 1 0.08 A120Ta E.4 1 0.08 I148T E.4 1 0.08 711+3A→Ta I.5 1 0.08 H199Y E.6a 1 0.08 875+1G→A I.6a 1 0.08 Table 1 (continued) Mutation Exon/intron CF alleles % 1717-1G→A I.10 1 0.08 L571S E.12 1 0.08 T582Ra E.12 1 0.08 E585X E.12 1 0.08 1898+3A→G I.12 1 0.08 G673X E.13 1 0.08 E692Xa E.13 1 0.08 R851X E.14a 1 0.08 R851La E.14a 1 0.08 A1006E E.17a 1 0.08 L1065Ra E.17b 1 0.08 F1074La E.17b 1 0.08 R1158X E.19 1 0.08 3667del4a E.19 1 0.08 3860ins31a E.20 1 0.08 3905insT E.20 1 0.08 4005+1G→A I.20 1 0.08 Q1281Xa E.20 1 0.08 Q1313X E.21 1 0.08 Known mutations (75) 1155 90.23 Unknown mutations 125 9.77 a Mutations discovered by the CF group of the Medical and Molecular Genetics Centre - IRO, Barcelona, Spain that range between 0.5% and 0.9%, representing 6.0% of the CF chromosomes. Login to comment

PMID: 16678395 [PubMed] Munthe-Kaas MC et al: "CFTR gene mutations and asthma in the Norwegian Environment and Childhood Asthma study."

No. Sentence Comment

25 CFTR mutation Alleles (%) F508del 184 (62.2) R117C 12 (4.1) R117H 12 394delTT 11 (3.8) 4005+2T-C 11 G551D 6 (2.0) 3659delC 5 (1.7) E60X 4 (1.4) V232D 4 1525-2A-G 3 (1.0) N1303K 3 G542X 2 (0.7) E279X 2 R75X 2 S912X 2 E116X 1 (0.3) L295Q 1 R347L 1 Q493X 1 I506L 1 I507del 1 R553X 1 G576A 1 621-1G-T 1 2183AA-G 1 S945L 1 R1162X 1 I1234V 1 3849+10 kbC-T 1 W1282X 1 Unknown 18 (6.5) Total alleles 296 (100%) Mutations detected with OLA31 m kit-74%. Login to comment

PMID: 16963320 [PubMed] Perez MM et al: "CFTR gene analysis in Latin American CF patients: heterogeneous origin and distribution of mutations across the continent."

No. Sentence Comment

42 Some have concentrated in the search of specific mutations that are Table 1 Mutations found in the Latin American CF patients Exon 1 p.L6VÌe; Exon 3 p.W57X, p.R75X, p.G85E Exon 4 p.R117H Exon 6a p.H199Y, p.V201M, p.L206W, p.Q220X, p.V232D, c.846delTÌe; Exon 6b p.Y275XÌe;, c.935delA Exon 7 p.R334W, p.R347P, p.Y362XÌe;, c.1078delT, c.1215delG Exon 8 c.1323_1324insAÌe; Exon 9 c.1460_1461delATÌe;, c.1353_1354insTÌe;,# Exon 10 p.I506T, p.I507del, p.F508del Exon 11 p.G542X, p.S549N, p.S549R, p.G551D, p.G551S, p.R553X, p.L558S, p.A559T, c.1782delA Exon 12 p.S589I Exon 13 p.H609RÌe;, p.P750L, p.V754M, c.1924_1930del, c.2055_2063del, c.2183AA NG;c.2184delA, c.2184delA, c.2185_2186insC, c.2347delG, c.2566_2567insTÌe;, c.2594_2595delGTÌe; Exon 14a p.R851L, c.2686_2687insTÌe; Exon 15 c.2869_2870insG Exon 16 c.3120+1GNA Exon 17a p.I1027T, c.3171delC, c.3199_3204del Exon 17b p.G1061R, p.R1066C, p.W1069X#, p.W1089X, p.Y1092X, p.W1098CÌe; Exon 19 p.R1162X, p.W1204X, p.Q1238X, c.3617_3618delGAÌe;#, c.3659delC Exon 20 p.W1282X, p.R1283M Exon 21 p.N1303K, c.4016_4017insT Exon 22 c.4160_4161insGGGGÌe; 5' flanking c.-834GNT Intron 2 c.297-1GNAÌe;, c.297-2ANG Intron 3 c.406-1GNA Intron 4 c.621+1GNT Intron 5 c.711+1GNT Intron 8 c.IVS8-5T Intron 10 c.1716GNA, c.1717-1GNA Intron 11 c.1811+1.6KbANG, c.1812-1GNA Intron 12 c.1898+1GNA, c.1898+3ANG Intron 14 c.2789+2_2789+3insA, c.2789+5GNA Intron 17a c.3272-26ANG Intron 17b c.3500-2ANGÌe; Intron 19 c.3849+1GNA, c.3849+10KbCNT Intron 20 c.4005+1GNA, c.4005-1GNA# Mutations are listed according to their position in the gene. Login to comment
46 of chromosomes analysed p.F508del p.G542X p.W1282X p.N1303K p.R1162X p.L6VÌe; p.W57X p.R75X p.G85E p.R117H p.H199Y p.V201M p.L206W p.Q220X p.V232D p.Y275XÌe; p.R334W p.R347P p.Y362XÌe; p.I506T Argentina 98 61 440 258 18 12 12 2 1 1 3 1 5 1 310 181 20 7 5 5 7 0 5 0 222 135 15 7 5 1 26 14 2 1 1 150 88 6 6 1 2 3 Subtotal and frequency (%) 1246 100 737 59.15 61 4.90 27 2.17 28 2.25 9 0.72 1 0.08 1 0.08 13 1.04 1 0.08 13 1.04 1 0.08 Brazil 468 221 26 11 74 38 2 1 320 155 28 3 8 8 4 1 2 1 1 8 122 62 120 38 10 3 148 38 4 0 0 48 15 154 75 5 1 0 2 0 386 154 24 6 10 17 9 0 10 1 18 4 0 0 2 0 0 0 0 Subtotal and frequency (%) 1858 100 800 43.06 99 5.33 11 0.59 34 1.83 25 1.35 13 0.70 1 0.05 2 0.11 1 0.05 1 0.05 20 1.07 1 0.05 Chile 72 21 36 11 3 0 44 22 4 3 1 1 100 45 7 5 0 2 0 2 0 Subtotal and frequency (%) 252 100 99 41.28 14 5.55 8 3.17 3 1.19 3 1.19 Colombia 184 77 7 2 1 2 1 34 13 2 1 1 Subtotal and frequency (%) 218 100 90 41.28 9 4.13 3 1.38 2 0.92 2 0.92 1 0.46 Costa Rica Frequency (%) 48 100 11 22.91 12 25.00 0 0 0 0 0 Cuba Frequency (%) 144 100 49 34.03 Ecuador 32 11 1 50 16 2 2 20 5 0 0 0 Subtotal and frequency (%) 102 100 32 31.37 2 1.96 1 0.98 2 1.96 Mexico 194 79 12 4 3 1 1 1 2 80 36 4 1 Subtotal and frequency (%) 274 100 115 41.97 16 5.84 5 1.82 3 1.09 1 0.36 1 0.36 1 0.36 2 0.73 Uruguay Frequency (%) 76 100 43 56.58 6 7.89 2 2.63 3 3.95 3 3.95 2 2.63 Venezuela 54 16 2 82 41 Subtotal and frequency (%) 136 100 57 41.91 2 1.47 Total 4354 2033 221 49 72 42 1 1 3 32 1 1 1 2 1 1 1 39 1 1 2 Frequency (%) 100 46.69 5.08 1.13 1.65 0.96 0.02 0.02 0.07 0.73 0.02 0.02 0.02 0.05 0.02 0.02 0.02 0.90 0.02 0.02 0.05 The five most frequent mutations are shown on the left-hand side, followed by the rest of the mutations in 5'-3' and exon-intron order. Login to comment
98 As an example, in the case of Argentina and Uruguay, the p.F508del mutation shows the highest frequencies (59% and Table 5 Mutations with frequencies less than 0.1% Panel A Mutation Number of chromosomes % Country p.R75X 3 0.07 Mexico c.W1089X 3 0.07 Argentina, Brazil c.406-1GNA 3 0.07 Mexico c.1898+1GNA 3 0.07 Argentina, Brazil c.2686_2687insTÌe; 3 0.07 Argentina, Brazil p.L206W 2 0.05 Brazil p.I506T 2 0.05 Mexico p.S589I 2 0.05 Argentina c.711+1GNT 2 0.05 Argentina c.935delA 2 0.05 Mexico c.2055_2063del 2 0.05 Mexico c.2347delG 2 0.05 Brazil c.2566_2567insTÌe; 2 0.05 Argentina c.2789+2_2789+3insA 2 0.05 Argentina c.3199_3204del 2 0.05 Mexico c.3272-26ANG 2 0.05 Argentina c.4016_4017insT 2 0.05 Argentina Panel B Mutation N % each Country p.L6VÌe;, p.W57X, p.Q220X, p.Y362XÌe;, p.I1027T, p.G1061R, p.R1283M, c.297-2ANG, c.1353_1354insTÌe;, c.1460_1461delATÌe;, c.1782delA, c.1898+3ANG, c.2184delA, c.2594_2595delGTÌe;, c.2869_2870insG, c.4005Ìe;1GNA, c.4005-1GNA# 17 0.02 Argentina p.R117H, p.H199Y, p.G551S, p.L558S, p.P750L, p.V754M, p.W1069X#, p.W1098CÌe;, p.W1204X, c.297-1GNAÌe;, c.846delTÌe;, c.1078delT, c.1716GNA, c.1924_1930del, c.4160_4161insGGGGÌe; 15 0.02 Mexico p.V201M, p.V232D, p.Y275XÌe;, p.R347P, p.R851L, p.Q1238X, c.3171delC, c.3617_3618delGAÌe;# 8 0.02 Brazil p.A559T, p.H609RÌe;, c.1215delG, c.1323_1324insAÌe;, c.2185_2186insC, c.3500-2ANGÌe;, c.3849+1GNA, 7 0.02 Colombia c.-834GNT 1 0.02 Uruguay The upper part (Panel A) shows the mutations found in more than one patient, whereas the lower part (Panel B) of the table shows all the mutations that are present only once in each country. Login to comment

PMID: 23055971 [PubMed] Molinski S et al: "Functional Rescue of F508del-CFTR Using Small Molecule Correctors."

No. Sentence Comment

172 Corr-4a has a mild correction effect of the F508del-CFTR (effective in the low &#b5;M range), and a nearly complete correction effect on the rare mutant V232D-CFTR (Caldwell et al., 2011). Login to comment

PMID: 23835419 [PubMed] Loo TW et al: "Corrector VX-809 stabilizes the first transmembrane domain of CFTR."

No. Sentence Comment

175 By contrast, other CFTR mutants defective in processing such as V232D and H1085R have half-lives similar to wild-type CFTR after rescue [14]. Login to comment
176 To test if other CFTR processing mutants could be rescued by TM6 mutations, arginine mutations were introduced at positions Ile338, Ser341, Ile344, Val345, Met348, and Thr351 of the V232D and H1085R processing mutants. Login to comment
177 None of the introduced arginines were found to promote maturation of V232D or H1085R. Login to comment
178 Examples of typical results obtained with the I344R or M348R mutations introduced into V232D or H1085R are shown in Fig. 5B. Login to comment
179 It was possible that the processing mutations in the TMDs (V232D (TMD1) or H1085R (TMD2)) cannot be rescued by a direct rescue approach using suppressor mutations. Login to comment
180 To test if the V232D or H1085R mutants could be rescued by suppressor mutations in other domains, suppressor mutations in NBD1 (I539T), the NBD1-TMD2 interface (V510D), or TMD2 (R1070W) (only V232D) locations were introduced into the mutants. Login to comment
196 RDR1 differs from VX-809 however, as we find that it does not rescue processing mutations in other domains such as V232D (TMD1) or H1085R (TMD2) (unpublished observations). Login to comment
220 None of the arginines introduced into CFTR processing mutants DF508, V232D, or H1085R promoted maturation. Login to comment
236 (B) Extracts of cells expressing wild-type CFTR or mutants V232D or H1085R with or without the I344R or M348R mutations were subjected to immunoblot analysis. Login to comment
237 (C) Extracts of cells expressing processing mutants DF508, V232D, or H1085R with or without the V510D, I539T, or R1070W suppressor mutations were subjected to immunoblot analysis. Login to comment

PMID: 23865422 [PubMed] Loo TW et al: "Bithiazole correctors rescue CFTR mutants by two different mechanisms."

No. Sentence Comment

17 VX-809 appears to rescue CFTR processing mutants through direct interactions with TMD1.12,18 Bithiazoles, however, appear to rescue CFTR processing mutants by a different mechanism because bithiazoles have an additive effect on maturation when used in combination with VX-809.15 Identification of the bithiazole rescue site in CFTR is controversial as there is evidence that these compounds bind to NBD218 or to the TMDs.9 Evidence that bithiazoles interact with the TMDs was that 4a inhibited cross-linking between cysteines introduced into TMD1 and TMD219 and that 4a appears to stabilize TMD2.20 In addition, it was found that bithiazoles enhanced core glycosylation of a truncation mutant that contained only the TMDs.19 By contrast, it was recently reported that bithiazoles must interact with NBD2 because ƊF508 CFTR missing NBD2 was not rescued with bithiazoles and in silico docking predicted the presence of a bithiazole-binding site in NBD2.18 To test whether rescue of other CFTR processing mutants with bithiazoles (see Figure 1 for structures) was mediated by the TMDs or NBD2, we tested if bithiazoles could rescue full-length or ƊNBD2 CFTR mutants that had processing mutations in the TMDs such as G126D in TM2,21 V232D in Received: July 3, 2013 Revised: July 17, 2013 Published: July 18, 2013 Rapid Report pubs.acs.org/biochemistry (c) 2013 American Chemical Society dx.doi.org/10.1021/bi4008758 | Biochemistry 2013, 52, 5161-5163 Terms of Use TM4,20 F337R in TM6,12 and S1141R in TM12 (see Figure 2A). Login to comment
18 Mutants G126D and V232D are naturally occurring CF mutants, whereas F337R and S1141R mutants were constructed to map the orientation of the TM segments.12 The mutants were expressed for 18 h with or without VX-809 or the 4a, 4d, or 15Jf bithiazole correctors. Login to comment
20 Figure 2B shows that full-length and ƊNBD2 forms of G126D, V232D, and S1141R could be efficiently rescued with all the correctors such that the mature protein was the major product (Figure 2C,D). Login to comment
25 The second bithiazole rescue mechanism appears to involve the TMDs because removal of NBD2 had little effect on bithiazole rescue of mutants G126D, V232D, and S1141R and mutation F337R in TM6 inhibited rescue of full-length CFTR with bithiazoles. Login to comment

PMID: 23924900 [PubMed] Ren HY et al: "VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1."

No. Sentence Comment

60 There are several CF-associated mutations in MSD1 that cause defects in CFTR processing and function: N-terminal tail (E56K and P67L), TM1 (E92K), TM2 (L206W), and TM4 (V232D) (Figure 4, A-E). Login to comment
62 In contrast, 5 bc;M VX-809 only partially restored folding and function to E92K and V232D (Figure 4, A and E). Login to comment
65 V232D-CFTR was the least responsive to VX-809, and higher concentrations of the compound did not restore function beyond the 25% of normal CFTR observed in the presence of 5 bc;M VX-809. Login to comment
107 V232D-CFTR biogenesis and function to normal levels (Caldwell et al., 2011), these data suggest that correctors such as VX-809 and Corr4a can act to selectively suppress folding defects in CFTR caused by different disease-related mutations in MSD1. Login to comment

PMID: 24412276 [PubMed] Loo TW et al: "The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds."

No. Sentence Comment

0 The cystic fibrosis V232D mutation inhibits CFTR maturation by disrupting a hydrophobic pocket rather than formation of aberrant interhelical hydrogen bonds Tip W. Loo a,b , David M. Clarke *,a,b a Department of Medicine, University of Toronto, Toronto, ON, Canada M5S 1A8 b Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8 1. Login to comment
15 It appears that processing Biochemical Pharmacology 88 (2014) 46-57 A R T I C L E I N F O Article history: Received 11 November 2013 Received in revised form 30 December 2013 Accepted 31 December 2013 Available online 9 January 2014 Keywords: CFTR V232D processing mutation Cystic fibrosis Processing mutations Packing of TM segments Correctors A B S T R A C T Processing mutations that inhibit folding and trafficking of CFTR are the main cause of cystic fibrosis. Login to comment
17 Previous studies on helix-loop-helix fragments of the V232D processing mutation suggested that its mechanism was to lock transmembrane (TM) segments 3 and 4 together by a non-native hydrogen bond (Asp232(TM4)/Gln207(TM3)). Login to comment
19 The rationale was that a V232N mutation should mimic V232D and a V232D/Q207A mutant should mature if the processing defect was caused by hydrogen bonds. Login to comment
21 The V232N mutation did not mimic V232D as V232N showed 40% maturation compared to 2% for V232D. Login to comment
23 The Q207L mutation did not rescue V232D because Q207L showed about 50% maturation in the presence of corrector VX-809 while V232D/Q207A could no longer be rescued. Login to comment
24 These results suggest that V232D inhibits maturation by disrupting a hydrophobic pocket between TM segments rather than forming a non-native hydrogen bond. Login to comment
25 Disulfide cross-linking analysis of cysteines W356C(TM6) and W1145C(TM12) suggest that the V232D mutation inhibits maturation by trapping CFTR as a partially folded intermediate. Login to comment
26 Since correctors can efficiently rescue V232D CFTR, the results suggest that hydrophilic processing mutations facing a hydrophobic pocket are good candidates for rescue with pharmacological chaperones. Login to comment
37 Although P-gp studies have demonstrated that formation of non-native hydrogen bonds between TM segments promotes folding [22,23], a study of the V232D (TM4) CF processing mutation predicted that hydrogen bonds in CFTR TM segments would have the opposite effect and inhibit folding [24]. Login to comment
38 Therien et al. [24] used helix-loop-helix fragments of the CFTR TM3/TM4 region to study the V232D (TM4) mutation and concluded that the aspartate residue formed a non-native hydrogen bond with Gln207 in TM3 to disrupt TM3/TM4 interactions. Login to comment
39 Based on the conclusion that V232D inhibited maturation by forming a non-native hydrogen bond with Gln207, it was proposed that over 75 other CF mutations in the TMDs may also inhibit maturation through non-native hydrogen bond formation between helices [24]. Login to comment
41 In this study, we used a mutational approach to test if the mechanism of the V232D processing defect was due to formation of a non-native hydrogen bond with Gln207 in full-length CFTR. Login to comment
42 The rationale was that the V232N mutation should be just as effective as V232D to inhibit CFTR maturation and mutation of Gln207 to nonpolar residues should rescue the V232D mutant if the processing defect was caused by Asp232/Gln207 hydrogen bonds. Login to comment
70 Disulfide cross-linking analysis To compare the effect of V232D on packing of the TM segments, double cysteine mutants were generated for disulfide cross-linking analysis. Login to comment
80 Results 3.1. Replacement of Gln207 in TM3 with nonpolar residues does not rescue V232D CFTR The 1480 amino acids of CFTR are organized into two TMDs, each containing six TM segments, two NBDs, and an R domain [3] (Fig. 1A). Login to comment
85 The V232D mutation is a useful model system to examine the effects of processing mutations and correctors on CFTR maturation because it severely inhibits maturation such that CFTR accumulates as immature core-glycosylated protein in the ER. Login to comment
86 The V232D mutant however, can be efficiently rescued with correctors to yield mature protein at the cell surface with activity similar to the wild-type protein [6]. Login to comment
87 A previous study [24] on TM3/TM4 helix-loop-helix fragments suggested that V232D (TM4) caused CFTR misfolding by forming a non-native hydrogen bond with Gln207 (TM3). Login to comment
88 It was reported that the V232D mutation caused a TM3/TM4 helix-loop-helix fragment to migrate with increased mobility on SDS-PAGE gels relative to a wild-type fragment unless Gln207 was replaced by an amino acid such as leucine that did not form hydrogen bonds. Login to comment
90 If V232D inhibited CFTR maturation by forming a non-native hydrogen bond with Gln207, then we predicted that V232D would no longer inhibit maturation if Gln207 were replaced with an amino acid that could not form a hydrogen bond. Login to comment
96 Accordingly, Gln207 in the V232D mutant was replaced with a small (alanine) or large (leucine) amino acid that does not form hydrogen bonds. Login to comment
97 The Q207L mutation had previously been reported to abolish hydrogen bond interactions with V232D in TM3/4 helix-loop-helix fragments [24]. Login to comment
100 The V232D, V232D/Q207A, V232D/Q207L, and V232D/Q207C mutants were then expressed in the presence or absence of corrector VX-809 to test for maturation. Login to comment
101 Corrector VX-809 was used because it efficiently rescues V232D CFTR [33]. Login to comment
102 Whole cell SDS extracts were then subjected to immunoblot analysis. Fig. 1B shows that V232D severely inhibits maturation as only the 170 kDa immature protein was detected. Login to comment
103 Mature CFTR however, was observed when V232D was expressed in the presence of VX-809. Login to comment
104 By contrast, none of the Gln207 mutations rescued V232D CFTR (Fig. 1B and C) as no mature CFTR was observed when mutants V232D/Q207A, V232D/Q207L, or V232D/Q207C were expressed in the presence or absence of VX-809 (Fig. 1B). Login to comment
105 These results suggest that the Q207X mutations likely had deleterious effects on mutant V232D, as the mutants could no longer be rescued with VX-809. Login to comment
107 Mutations to Gln207 inhibit CFTR maturation Since V232D but not mutants V232D/Q207A, V232D/Q207L, or V232D/Q207C could be rescued with VX-809 (Fig. 1B), we tested if maturation of CFTR was also sensitive to changes to Gln207 in a wild-type background. Login to comment
109 Mutants V232D/Q207A, L, or C do not mature. Login to comment
112 (B) Whole cell extracts of cells expressing V232D or V232D/Q207A, L, or C mutants in the absence () or presence (+) of VX-809 were subjected to immunoblot analysis. Login to comment
116 An asterisk indicates significant (P < 0.05) inhibition when compared to V232D CFTR expressed in the presence of VX-809. Login to comment
125 The results suggest that Gln207 mutations might have an additive or synergistic effect on the V232D mutation. Login to comment
127 Inhibition of CFTR maturation correlates with the polarity of a Val232 mutation If V232D inhibits CFTR maturation by forming a non-native hydrogen bond with Gln207, then it would be expected that V232N and V232Q mutations would also severely inhibit maturation since asparagine and glutamine are structurally similar to aspartate and their amide groups can accept or donate two hydrogen bonds. Login to comment
130 The mutants were expressed in HEK 293 cells and whole cell extracts subjected to immunoblot analysis. Fig. 3A and B shows that the expression of mutant V232E resembled that of V232D as it significantly inhibited maturation by about 40-fold (about 2% of the CFTR protein was present as the mature protein when compared to 80% for wild-type CFTR (Fig. 3)). Login to comment
131 By contrast, the amount of mature protein was about 20-fold higher (about 40%) for mutants V232N and V232Q compared to V232D or V232E (Fig. 3). Login to comment
132 These results suggested that charge rather than non-native hydrogen bonds might be responsible for the severe reduction in CFTR maturation caused by the V232D or V232E mutations. Login to comment
157 The results suggest that the V232D mutation may severely inhibit maturation because it introduces charge into a relatively hydrophobic region in the TM segments. Login to comment
159 3.4. Rescue of Val232 processing mutants with correctors The V232D CFTR could be efficiently rescued with either VX-809 [19] or bithiazoles [33]. Login to comment
169 For example, V510D promotes maturation of mutants with processing mutations in TMD1 (V232D), TMD2 (H1085R) and NBD1 (DF508) whereas other suppressors such as I539T and R1070W promote maturation of DF508 CFTR but not mutants V232D or H1085R [19]. Login to comment
178 The properties of the V232E mutant are likely to be very similar to the V232D mutant as the V510D suppressor mutation [19] could also rescue the V232D mutant. Login to comment
195 The L305R(TM5) P-gp mutant resembles CFTR mutants V232D, V232E, and V232K The characteristics of the V232D/Q207X (Fig. 1) and V232X (Fig. 3) mutants suggest that the mechanism of the V232D mutation involves disruption of a hydrophobic pocket between TM segments. Login to comment
204 It appears that the CFTR V232D, V232E, and V232K processing mutationsresemble P-gp mutantsthatcontain acharged residueata hydrophobic interface between adjacent TM segments because they can be efficiently rescued (with VX-809). Login to comment
241 We predict that the V232D CFTR mutant might be similar to L305R P-gp because it involves insertion of a charged residue into a hydrophobic pocket. Login to comment
258 Cross-linking analysis suggests that V232D causes incomplete packing of the TM segments Previous studies on processing mutations in NBD1 (DF508) or in the fourth intracellular loop connecting TM segments 10 and 11 (Q1071P or H1085R) showed that they trapped CFTR at an early folding step resulting in incomplete packing of the TM segments [17,50]. Login to comment
263 To test if V232D trapped CFTR in a conformation with incomplete packing of the TM segments, the mutation was introduced into a Cys-less CFTR containing W356C(TM6) and W1145C(TM12). Login to comment
264 Cys-less CFTR, mutants W356C/W1145C and V232D/W356C/W1145C were expressed in HEK 293 cells in the absence or presence of 5 mM VX-809. Membranes were prepared Fig. 8. Login to comment
271 No cross-linked product was detected in Cys-less CFTR or in mutant V232D/W356C/W1145C when expressed in the absence of corrector VX-809 (Fig. 10A and B). Login to comment
272 Expression of mutant V232D/W356C/W1145C in the presence of corrector however, promoted maturation of the protein such it could be efficiently cross-linked with either M8M or BMH (about 80-85% cross-linked product; Fig. 10B). Login to comment
273 These results suggest that the V232D mutation traps CFTR in a partially folded conformation with incomplete packing of the TM segments that can be corrected with VX-809. Login to comment
274 In summary, the results suggest that: (1) the mechanism of how V232D causes protein misfolding is unlikely to involve non-native hydrogen bond interactions with Gln207 because V232N yielded about 20-fold more mature CFTR than V232D; (2) it appears that the mechanism of protein misfolding by V232D mutation involves disruption of a hydrophobic pocket since the hydrophobicity of the substituted amino acid at position 232 correlated with the amount of mature product and the ability to correct the defects with correctors or the V510D suppressor mutation; (3) the V232D mutation traps CFTR as a partially folded intermediate that can be rescued by corrector VX-809 to yield a native structure. Login to comment
276 Discussion The results in this study suggest that the mechanism for inhibition of packing of TM segments caused by the V232D mutation may be different in the full-length protein than predicted by the TM3/4 helix-loop-helix model system. Login to comment
277 Therien et al. [24] studied the V232D mutation in the TM3/4 helix-loop-helix fragment and suggested that the fragment was a useful model system for studying the mechanism of the V232D processing mutation because most consecutive TM segments in membrane proteins were in contact with one another [51]. Login to comment
278 In the TM3/4 helix-loop-helix model system, it was predicted that the two TM segments would be adjacent to each other along their entire lengths to bring Gln207(TM3) in close contact with TM4 to form a non-native hydrogen bond with the V232D processing mutation. Login to comment
279 It was postulated that the V232D mutation formed a hydrogen bond with Gln207 because replacement of Gln207 with nonpolar residues reversed the abnormal migration pattern of the V232D TM3/4 fragment in SDS-PAGE gels. Login to comment
280 They showed that the migration pattern of mutant V232D/Q207L in SDS-PAGE gels resembled that of the wild-type fragment. Login to comment
281 The results of TM3/4 helix-loop-helix model system studies were used to predict that hydrogen bond formation between V232D and Gln207 could alter the normal assembly and alignment of CFTR TM segments in full-length CFTR. Login to comment
282 Our studies on the full-length CFTR protein however, suggest that V232D inhibited maturation by disrupting a hydrophobic pocket. Login to comment
284 Although mutants such as A207A, Q207L and Q207C could be rescued with corrector VX-809 (Fig. 2), the V232D mutation appeared to have an effect that was independent of that of Gln207 since mutants Q207A/V232D, Q207L/V232D and Q207C/V232D could no longer be rescued by VX-809 (Fig. 1B). Login to comment
292 The V232D mutation inhibits packing of the TM segments. Login to comment
293 (A) Membranes prepared from cells expressing mutants W356C/W1145C, V232D/W356C/W1145C or Cys-less CFTR in the absence (none) or presence of corrector VX-809 were treated without (none) or with cross-linkers (X-linkers) M8M or BMH for 10 min at 20 8C. Login to comment
301 The P-gp L305R mutation in TM5 resembles the CFTR V232D mutation because it also inhibits maturation by disrupting a TM4/5 hydrophobic interface (Fig. 7A). Login to comment
313 We predict that V232D inhibits CFTR maturation by a mechanism that is similar to that proposed for the Q1071P, H1085R [50] and DF508 [17,26] processing mutations (see Fig. 11). Login to comment
317 The V232D mutation traps CFTR at an early folding step with incomplete packing of the TM segments and incomplete domain assembly (Fig. 11B). Login to comment
321 Corrector 4a may also efficiently rescue V232D CFTR because it also appears to interact with the transmembrane domains [18,65]. Login to comment
323 In summary, mutational analysis suggests that the mechanism of protein misfolding by the V232D mutation in TM4 does not involve non-native hydrogen bond formation with Gln207 in TM3. Login to comment
325 Understanding the mechanism of protein misfolding by V232D CFTR and its rescue by correctors is important because more than 50 other CF mutations have been identified in the TM segments of CFTR that involve a change from nonpolar to polar/charged residue [66]. Login to comment
327 Model of inhibition of CFTR by V232D and rescue by corrector VX-809. Login to comment
330 (B) The presence of V232D (red) in TM4 disrupts the hydrophobic environment and inhibits proper folding to trap the mutant protein as a partially folded intermediate. Login to comment
332 (C) Studies [19,20] suggest that corrector VX-809 interacts with TMD1 to induce V232D to complete the folding process to yield a native structure in which W356C and W1145C can be cross-linked (orange line). Login to comment

PMID: 25381027 [PubMed] Collawn JF et al: "CFTR and lung homeostasis."

No. Sentence Comment

130 V232D is a rare folding mutation that can be corrected to near WT, maturely glycosylated protein levels with small molecule corrector Corr-4a (11). Login to comment

PMID: 25697318 [PubMed] Lay-Son R G et al: "[CFTR gene sequencing in a group of Chilean patients with cystic fibrosis]."

No. Sentence Comment

58 Mutaciones detectadas por secuenciaci&#f3;n masiva en cohorte de 39 pacientes chilenos con FQ portadores de un alelo desconocido Mutaci&#f3;n detectada (nomenclatura actual*) n de alelos Reporte en pacientes con FQ (no de alelos) Efecto Denominaci&#f3;n antigua c.1330_1331delAT 3 Argentina (1)a Prote&#ed;na truncada por generaci&#f3;n de cod&#f3;n de t&#e9;rmino 1460delAT c.314T>A 2 Francia (1)a Cambio de amino&#e1;cido Isoleucina por Asparagina I105N c.4046G>A 2 Italia (7)b,c , EEUU (1)d Cambio de amino&#e1;cido Glicina por Aspartato G1349D c.148T>C 2 Espa&#f1;a (2)e Cambio de amino&#e1;cido Serina por Prolina S50P c.695T>A 1 Espa&#f1;a (14)e,f , EEUU (hispanos) (5)g,h Francia (2)a , Brasil (1)i Cambio de amino&#e1;cido Valina por Aspartato V232D c.3266G>A 1 Espa&#f1;a (5)e , Brasil (2)i,j , EEUU (hispanos) (2)g , Argentina (1)k , Israel (1)l Prote&#ed;na truncada por generaci&#f3;n de cod&#f3;n de t&#e9;rmino W1089X c.1647T>G 1 Emiratos &#c1;rabes Unidos (> 30)m,n , Colombia (4)o , Israel (4)p , Argelia (2)p , Marruecos (2)q , Reino Unido (2)p , Portugal (1)p , Espa&#f1;a (1)p , Francia (1)p , Italia (1)p , Brasil (1)q , Argentina (1)q Cambio de amino&#e1;cido Serina por Arginina S549R(T- >G) c.308G>A 1 No descrita previamente Cambio de amino&#e1;cido Glicina por Glutamato G103E c.1680-1G>A 1 Espa&#f1;a (1)r Alteraci&#f3;n en splicing 1812-1G->A c.1679+1G>C 1 Francia (2)s Macedonia (1)s , Alteraci&#f3;n en splicing 1811+1G->C c.490-2A>G 1 Argentina (1)t Alteraci&#f3;n en splicing 622-2A->G FQ: Fibrosis qu&#ed;stica. Login to comment