ABCMdb

PMID: 18306312

Gene GG, Llobet A, Larriba S, de Semir D, Martinez I, Escalada A, Solsona C, Casals T, Aran JM
N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel.
Hum Mutat. 2008 May;29(5):738-49., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Gly85Glu ABCC7 p.Arg75Gln ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Tyr89Cys ABCC7 p.Glu60Lys By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Login to comment 4 ABCC7 p.Gly85Glu ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Glu60Lys Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Login to comment 6 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Login to comment 8 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Login to comment 9 ABCC7 p.Arg75Gln Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology. Login to comment 40 ABCC7 p.Gly85Glu ABCC7 p.Arg75Gln ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Tyr89Cys ABCC7 p.Gly85Val The eight CFTR variants included in this study: p.P5L, p.S50P, p.E60 K, p.R75Q, p.G85E, p.G85V, p.Y89C, and p.E92K (Fig. 1A) were generated by oligonucleotide-directed mutagenesis in pCMVCFTRNot6.2wt using the QuickChangeTM XL-Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer`s instructions (see Supplementary Table S1 for a detailed description of the mutagenesis primers employed; available online at http://www.interscience.wiley.com/jpages/1059-7794/supp mat). Login to comment 76 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys The average capacitances of HEK 293 cells transfected with wild-type CFTR and each of the three active channel variants were: wild-type CFTR (1671 pF; n 5 24); p.R75Q (2473 pF; n 5 18); p.Y89C (2072 pF; n 5 6); and p.P5L (2173 pF; n 5 20). Login to comment 102 ABCC7 p.Gly85Glu ABCC7 p.Arg75Gln ABCC7 p.Ser50Pro ABCC7 p.Tyr89Cys ABCC7 p.Glu60Lys ABCC7 p.Gly85Val RESULTS Description and Cross-Species Analysis of Natural N-Terminus CFTR Variants We chose eight naturally occurring sequence variants, four located across the N-terminal CFTR tail (p.P5L, p.S50P, p.E60K, and p.R75Q), and four within the first segment of MSD1 (p.G85E, p.G85V, p.Y89C, and p.E92 K) (Fig. 1A; Table 1). Login to comment 109 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys Some of the variants such as p.R75Q and p.Y89C, presented a glycosylation status comparable to the wild-type CFTR, suggesting that they might reach the PM. Login to comment 110 ABCC7 p.Gly85Glu ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Glu60Lys ABCC7 p.Gly85Val In contrast, variants p.S50P, p.E60K, p.G85E, p.G85V, and p.E92K, produced only higher mobility band B proteins suggesting that, like the p.F508del mutant, the resulting misfolded channels are retained and degraded in the cytoplasm (Fig. 2A). Login to comment 116 ABCC7 p.Gly85Glu ABCC7 p.Arg75Gln ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Tyr89Cys ABCC7 p.Glu60Lys Moreover, some nascent/immature CFTR protein present in the ER and A B 501 E92K Y89C G85E/V P5L S50P E60K R75Q NBD1 NBD2 R FIGURE 1. Login to comment 118 ABCC7 p.Gly85Glu ABCC7 p.Arg75Gln ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Tyr89Cys ABCC7 p.Glu60Lys ABCC7 p.Gly85Val B: Alignment of the N-terminus (amino acids 1 to 100) of the CFTR protein derived from di¡erent species.The sequences derived from human (Homo sapiens, Gen- BankNM_000492), mouse (Mus musculus,GenBankNM_021050), Norway rat (Rattusnovergicus,GenBankNM_031506), European rabbit (Oryctolagus cuniculus, GenBank NM_001082716), cow (Bos taurus, GenBank NM_174018), sheep (Ovis aries, GenBank NM_001009781), African clawed frog (Xenopus laevis, GenBank X65256), and spiny dog'sh (Squalus acanthias, GenBank M83785) were aligned using the ClustalW multiple sequence alignment program.The amino acid residue a¡ected by each of the variants analyzed (p.P5L, p.S50P, p.E60K, p.R75Q, p.G85E, p.G85V, p.Y89C, and p.E92K) is indicated in bold. Login to comment 120 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys Likewise, variants p.R75Q and p.Y89C (Fig. 3) presented a localization pattern similar to that of wild-type CFTR, with most of the protein visualized in the PM (yellow colocalization of the merge images). Login to comment 122 ABCC7 p.Gly85Glu ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Glu60Lys ABCC7 p.Gly85Val Similarly, variants p.S50P, p.E60K, p.G85V, p.G85E, and p.E92K displayed a yellow colocalization pattern clearly compatible with retention of the anomalous CFTR proteins within the intracellular compartments and no detection of PM staining (Fig. 3). Login to comment 130 ABCC7 p.Gly85Glu ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Glu60Lys ABCC7 p.Gly85Val Likewise, none of the five variants (p.S50P, p.E60K, p.G85E, p.G85V, and p.E92K) in which severe misprocessing was previously demonstrated (Figs. 2 and 3), was able to generate cAMP-activated currents (Fig. 4B). Login to comment 131 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys From the variants expressed in the PM (Fig. 3), only p.R75Q and p.Y89C generated cAMP-stimulated currents comparable to that of wild-type CFTR channel (Fig. 4C). Login to comment 133 ABCC7 p.Arg347His ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Gly542* ABCC7 p.Pro205Ser ABCC7 p.Leu206Trp ABCC7 p.Gly85Glu ABCC7 p.Gly85Glu ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Tyr1092* ABCC7 p.Gly576Ala ABCC7 p.Leu997Phe ABCC7 p.Gln890* ABCC7 p.Ala1006Glu ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Glu725Lys ABCC7 p.Tyr89Cys ABCC7 p.Glu60Lys ABCC7 p.Gly85Val Genotype^Phenotype Correlation in the N-Terminal CFTR MissenseVariants Under StudyÃ Missense varianta Phenotype Second allele (number of patients)b p.P5L CF p.F508del (1), p.P205S (1) p.S50P CBAVD p.F508del (1), p.E115del (1) p.E60K CF p.G542X (1), p.I507del (1) p.R75Q HT p.F508del (3), p.E725K (1) B p.R347H (1), p.R75Q (1), n.i. (4) Br c.1584G4A (2), c.1210-7_1210-6delTT (1), n.i.(3) NT p.F508del (1) CP c.1584G4A (1), n.i. (3) MI n.i. (1) CUAVD n.i. (2) OZ n.i. (2) Normal p.R75Q (1), c.2052_2053insA (1), n.i. (1) p.G85E CF p.F508del (8), p.G542X (2), p.I507del (1), c.580-1G4T (1), p.G85E (1), c.1477_ 1478delCA (1) CBAVD p.G576A (1) HT p.L997F (1),WT (1) p.G85V CF p.F508del (2), p.G542X (2), p.Y1092X (1), c.265715G4A (1), p.A1006E, c.1210-7_1210- 6delTT (1), n.i. (1) p.Y89C CF n.i. (1)c p.E92K CF p.F508del (2), p.Q890X (1), p.L206W (1) CBAVD c.1210-7_1210-6delTT (1) ÃThe recommendations for mutation nomenclature (www.hgvs.org/mutnomen/) were used to name CFTR gene sequence variations at both the nucleotide level and the protein level. Login to comment 136 ABCC7 p.Gly85Glu ABCC7 p.Glu92Lys Besides being present in CF patients, we have found both p.G85E and p.E92K variants in CBAVD patients [Casals et al., 2000]. Login to comment 137 ABCC7 p.Gly85Glu Moreover, the p.G85E variant was also detected in one newborn with hypertrypsinemia [Gartner et al., 2003]. Login to comment 138 ABCC7 p.Arg75Gln ABCC7 p.Ser50Pro The p.S50P variant was only found in CBAVD patients [Casals et al., 2000], whereas the p.R75Q variant was described as a neutral change (CFMDB) and reported later in several CFTR-related disorders such as obstructive azoospermia [Dork et al., 1997], bronchiectasis [Casals et al., 2004b], chronic pancreatitis [Casals et al., 2004a], hypertrypsinemia [Gartner et al., 2003], as well as in the general population [Bombieri et al., 2000]. Login to comment 151 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys Similar values were observed for p.R75Q (98755 pA/pF, n 5 18), and p.Y89C (114754 pA/pF, n 5 6), indicating that these CFTR variants were trafficked to the PM. Login to comment 156 ABCC7 p.Gly85Glu ABCC7 p.Gly85Glu ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Ser50Pro ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Glu92Lys ABCC7 p.Tyr89Cys ABCC7 p.Tyr89Cys ABCC7 p.Glu60Lys ABCC7 p.Glu60Lys ABCC7 p.Gly85Val ABCC7 p.Gly85Val Confocal images from representative xy sections taken from1of 3 independent experiments show the subcellular distribution of wild-type CFTR (WT), p.F508del mutant (F508del), and variants p.S50P (S50P), p.E60K (E60K), p.G85E (G85E), p.G85V (G85V), p.E92K (E92K), p.P5L (P5L), p.R75Q (R75Q), and p.Y89C (Y89C). Login to comment 158 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys In the £uorescent images from variants p.P5L, p.R75Q, and p.Y89C, the contour plot from a successfully transfected cell, obtained from the corresponding phase contrast image, is shown to aid the identi'cation of the cell boundary. Login to comment 166 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys Single-Channel Analysis of N-Terminus CFTR Variants Expressed at the Plasma Membrane To further investigate whether variants p.R75Q, and p.Y89C affected the chloride channel function of CFTR and, particularly, to delineate whether the reduced current density of p.P5L could be attributed only to a folding/trafficking defect or also to a channel malfunction, single-channel experiments were carried out. Login to comment 180 ABCC7 p.Gly85Glu ABCC7 p.Gly85Glu ABCC7 p.Ser50Pro ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Glu92Lys ABCC7 p.Glu60Lys ABCC7 p.Glu60Lys ABCC7 p.Gly85Val ABCC7 p.Gly85Val B: Recordings fromvariants p.S50P (S50P), p.E60K (E60K), p.G85E (G85E), p.G85V (G85V), and p.E92K (E92K) (superimposed recordings). Login to comment 182 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys ABCC7 p.Tyr89Cys C:Whole-cell currents evoked by variants p. R75Q (R75Q), p.P5L (P5L), and p.Y89C (Y89C).Wild-type CFTR current (WT) is also shown for comparison. Forskolin was applied for 90s. Login to comment 183 ABCC7 p.Arg75Gln D:Tra/cking of p.R75Q and p.P5L variants to the PM. Login to comment 184 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln Current densities of wild-type CFTR (WT), p.R75Q (R75Q) and p.P5L (P5L) variants depended on the posttransfection time. Login to comment 197 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys As explained above, variants p.R75Q and p.Y89C were also able to reach the PM and supported chloride currents (Fig. 4). Login to comment 198 ABCC7 p.Arg75Gln Single-channel records showed that variant p.R75Q displayed a phenotype similar to wild-type, in terms of NPo (0.3070.07), to (2967116 ms), and chord conductance (8.670.8 pS) (n 5 10) (Fig. 5; Table 2). Login to comment 199 ABCC7 p.Tyr89Cys Finally, the p.Y89C CFTR was at some extent, FIGURE 5. Login to comment 200 ABCC7 p.Arg75Gln Representative single-channel recordings of wild-type CFTR, and variants p. R75Q and p.P5L. Login to comment 201 ABCC7 p.Arg75Gln ABCC7 p.Arg75Gln A: Single channel records showing the behavior of wild-type CFTR (WT), p.R75Q (R75Q), and p.P5L (P5L) variants over a prolonged period. Login to comment 205 ABCC7 p.Arg75Gln Summary of Single-Channel Analysis From N-Terminal CFTR Variantsy Variant (number of patches) NPo to (ms) G (pS) Wild-type (n 511) 0.3070.05 259754 8.670.6 p.P5L (n 5 7) 0.0970.05Ã 77715Ã 4.870.3Ã p.R75Q (n 510) 0.3070.07 2967116 8.670.8 y Summary of single-channel electrophysiological properties of the N-terminal CFTR variants studied that showed successful tra/cking to the plasma membrane. Login to comment 213 ABCC7 p.Gly85Glu ABCC7 p.Arg75Gln ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Tyr89Cys ABCC7 p.Gly85Val Four of the variants (p.P5L, p.S50P, p.E60 K, and p.R75Q) are localized within the cytosolic N-terminal tail, and the remaining four (p.G85E, p.G85V, p.Y89C, and p.E92K) are embedded in three positions within the first transmembrane segment (TM1) of MSD1. Login to comment 215 ABCC7 p.Gly85Glu ABCC7 p.Ser50Pro ABCC7 p.Glu92Lys ABCC7 p.Glu60Lys ABCC7 p.Gly85Val Accordingly, using three different approaches (immunoblotting, immunocytochemistry, and electrophysiology) we found that 5 (p.S50P, p.E60K, p.G85E, p.G85V, and p.E92K) out of the 8 variants failed to mature, showing an analogous behavior than the most common F508del mutation. Login to comment 218 ABCC7 p.Arg75Gln ABCC7 p.Tyr89Cys Conversely, we have observed that three CFTR missense variants, two (p.P5L and p.R75Q) located in the cytosolic N-terminus and one (p.Y89C) located within the first segment of MSD1, appeared to mature and reach the PM, albeit with different efficiencies. Login to comment 219 ABCC7 p.Gly85Glu MSD1-A¡ectingVariants Concerning the p.G85E mutation, our results are in agreement with those reported by Decaestecker et al. [2004] through pulse-chase experiments. Login to comment 220 ABCC7 p.Gly85Glu ABCC7 p.Glu92Lys ABCC7 p.Gly85Val Moreover, although both p.G85E and p.G85V, similarly to p.E92K, do not appear to affect TM1 topology [Xiong et al., 1997], they are also localized within or adjacent to the bilayer. Login to comment 222 ABCC7 p.Gly85Glu Indeed, it has been recently shown that Derlin-1, an ERAD component, efficiently degrades p.G85E and p.F508del folding mutants [Sun et al., 2006]. Login to comment 224 ABCC7 p.Glu92Lys Variant p.E92K results in an amino acid of opposite charge with potential consequences in CFTR folding. Login to comment 228 ABCC7 p.Gly85Glu ABCC7 p.Glu92Lys ABCC7 p.Tyr89Cys Regarding the p.Y89C variant, based on the structural features of the amino acids involved (the bulky hydrophobic tyrosine residue is substituted by the smaller hydrophilic cysteine residue) and its position within TM1 (between the severe folding mutations p.G85E/V and p.E92K) it would also be expected a major structural rearrangement and folding defect of MSD1 conferred by this variant. Login to comment 229 ABCC7 p.Tyr89Cys Surprisingly, p.Y89C CFTR was able to mature quite efficiently and to reach the PM. Login to comment 232 ABCC7 p.Tyr89Cys Nevertheless, additional work is necessary to accurately define the electrophysiological properties of the p.Y89C variant. Login to comment 236 ABCC7 p.Ser50Pro ABCC7 p.Glu60Lys Thus, all these important interactions might be perturbed by the N-tail CFTR folding mutations p.S50P and p.E60K. Login to comment 237 ABCC7 p.Ser50Pro In the former, proline residues, such as the introduced in the p.S50P mutation, are not favored, introducing ''kinks`` in a-helices because the backbone nitrogen is not available for hydrogen bonding and because of steric constraints caused by their ring structure [Richardson and Richardson, 1989]. Login to comment 238 ABCC7 p.Glu60Lys Conversely, p.E60K results in an amino acid of opposite charge. Login to comment 240 ABCC7 p.Arg75Gln For the remaining N-terminus CFTR cytosolic variants showing maturation and PM localization, p.R75Q also affected the charge of the position as a result of amino acid exchange. Login to comment 241 ABCC7 p.Arg75Gln Nevertheless, p.R75Q CFTR both displayed a maturation pattern and subcellular localization analogous to the wild-type CFTR, and did not cause major alterations in intrinsic chloride channel activity. Login to comment 242 ABCC7 p.Arg75Gln Thus, the fact that p.R75Q is located at the hinge between the N-terminal CFTR cytosolic tail and the first segment of MSD1 may involve some degree of structural interdomain flexibility. Login to comment 243 ABCC7 p.Arg75Gln Our data seem to corroborate previous results from genotype-clinical phenotype correlations [Divac et al., 2004; Ravnik-Glavac et al., 2000; Ziedalski et al., 2006], leading to the p.R75Q variant`s consideration as a polymorphism [Nikolic et al., 2006]. Login to comment 245 ABCC7 p.Ser50Pro ABCC7 p.Glu60Lys Indeed, similarly to the above-referred cytosolic N-terminal CFTR variants p.S50P and p.E60K, folding and/or trafficking/processing defects seem to be the major factors contributing to the abnormal p.P5L CFTR phenotype, which showed a greatly reduced expression at the PM by immunodetection. Login to comment 247 ABCC7 p.Arg31Cys ABCC7 p.Arg31Leu A recent report analyzing the natural mutations p.R31C and p.R31L, has shown that these subtle N-terminus alterations compromise biogenesis and enhance internalization of CFTR, contributing to the loss of surface expression and the associated defect in chloride conductance of the channel [Jurkuvenaite et al., 2006]. Login to comment 255 ABCC7 p.Tyr89Cys Genotype^Phenotype Correlations All selected N-terminus CFTR missense variants except p.Y89C, described in an Italian patient [Padoan et al., 2000], have been identified in Spanish individuals with CF or different CFTR-related disorders (see Table 1 and references therein). Login to comment 257 ABCC7 p.Ser50Pro The main exception is variant p.S50P, which, despite exhibiting absence of mature CFTR protein, was merely found in two CBAVD patients [Casals et al., 2000]. Login to comment