ABCMdb

PMID: 18769034

Grangeia A, Barro-Soria R, Carvalho F, Damas AM, Mauricio AC, Kunzelmann K, Barros A, Sousa M
Molecular and functional characterization of CBAVD-causing mutations located in CFTR nucleotide-binding domains.
Cell Physiol Biochem. 2008;22(1-4):79-92. Epub 2008 Jul 25., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser A previous screening of the entire coding region of the cystic fibrosis transmembrane conductance regulator gene (CFTR [MIM 602421]) in CBAVD patients identified three novel mutations: P439S is located in the first nucleotide binding domain (NBD1) of CFTR, whereas P1290S and E1401K are located in NBD2. Login to comment 4 ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Results: Although maturation patterns were not affected, total amounts of mature P439S-CFTR and P1290S-CFTR were reduced. Login to comment 5 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Confocal microscopy showed correct membrane localisation of E1401K-CFTR, whereas P439S-CFTR and P1290S-CFTR mutants were located mainly in the cytoplasm. Login to comment 7 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Conclusion: Dysfunction of CFTR is caused by either defective CFTR trafficking (P439S and P1290S) or/and Cl-channel function (P1290S and E1401K). Login to comment 59 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Plasmids, cell culture, transient transfection CFTR mutations (P439S, P1290S and E1401K) were generated (QuikChange site-directed mutagenesis kit, Stratagene, La Jolla, CA) in the eukaryotic expression vector pCMVCFTRNot6.2, according to the manufacturer`s instructions. Login to comment 97 ABCC7 p.Pro439Ser The P439S mutation was evaluated using three-dimensional structures from MJ0796 dimer (PDBid: 1L2T), F508del-CFTR hNBD1 (PDBid 1XMJ), and CFTR mNBD1 (PDBid: ROZ). Login to comment 102 ABCC7 p.Arg334Trp ABCC7 p.Pro439Ser P439S (NBD1) was identified in a 34 year old patient who carried the R334W mutation on the other chromosome. Login to comment 103 ABCC7 p.Pro1290Ser P1290S (NBD2) was identified in a 45 year old patient heterozygous for the Table 1. Login to comment 112 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Multiple alignments of CFTR amino acid sequences from different species and localization of NBD1, NBD2 and three novel CFTR mutations (P439S, P1290S, E1401K). Login to comment 123 ABCC7 p.Glu1401Lys E1401K (NBD2) was found in a 37 year old patient who carried the severe F508del mutation on the other chromosome. Login to comment 126 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Biosynthesis of CFTR mutants Maturation patterns of wild-type (wt) CFTR and mutant CFTR (F508del-CFTR, P439S-CFTR, P1290S-CFTR and E1401K-CFTR) were compared by Western blotting. Login to comment 130 ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser However, total amounts of mature P439S-CFTR and P1290S-CFTR were significantly reduced compared to wtCFTR, as indicated by densitometric analysis (Fig. 2B). Login to comment 137 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser The mutants P439S-CFTR and P1290S-CFTR were mainly expressed in the cytoplasm with little membrane expression, while E1401K-CFTR was clearly present in the cell membrane, similar to wtCFTR (Fig. 3A-B). Login to comment 142 ABCC7 p.Glu1401Lys wtCFTR and E1401K-CFTR were found in the plasma membrane, whereas F508del-CFTR is trapped in the cytoplasm. Login to comment 143 ABCC7 p.Pro1290Ser P439S-CFTR and P1290S-CFTR are detected predominantly in the cytoplasm but to some degree also in the plasma membrane. Login to comment 159 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Intracellular cAMP was enhanced by stimulation of the cells with 2 µM FSK and 100 µM IBMX, which largely increased the rate of I- influx in HEK293 cells expressing wtCFTR, while non-transfected cells or F508del-CFTR expressing cellsdidnotrespondtostimulation(Fig.4A-B).Expression of all three mutants P439S-CFTR, P1290S-CFTR and E1401K-CFTR allowed for cAMP-induced increase in I- influx, although the rate of I- influx was significantly lower than that for wtCFTR (Fig. 4A-B). Login to comment 160 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Reduced I- influx seems to be in accordance with the reduced levels of protein expression for two mutants P439S-CFTR, P1290S-CFTR.Although E1401K did not interfere with Fig. 5. Login to comment 165 ABCC7 p.Glu1401Lys ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser ABCC7 p.Pro439Ser GFP = green fluorescence protein, wt = wtCFTR, F508del = F508del-CFTR, P439S = P439S-CFTR, P1290S = P1290S-CFTR, E1401K = E1401K-CFTR. Login to comment 168 ABCC7 p.Pro1290Ser C) Effect of IBMX (100 µM) and forskolin (2 µM) on whole-cell currents of non-transfected cells and in cells expressing wtCFTR, F508del-CFTR and P1290S-CFTR. Login to comment 169 ABCC7 p.Pro1290Ser Current-voltage curves where mean whole-cell currents (in nA) were plotted against the Vc values (in mV), in non-transfected (black trace) and wtCFTR (red trace) expressing cells (left panel), and in cells expressing F508del-CFTR (black trace) or P1290S-CFTR (blue trace) (right panel). Login to comment 178 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Similar to the results from I- uptake studies, CFTR mutants P439S-CFTR, P1290S-CFTR and E1401K-CFTR showed a reduced but significant current increase and activation of whole cell conductance. Login to comment 180 ABCC7 p.Pro1290Ser Figure 5C represents current-voltage (IV) curves, where mean whole-cell currents (in nA) were plotted against the Vc values (in mV), in non-transfected cells and HEK293 cells expressing wtCFTR (left panel), and in HEK293 cells expressing F508del-CFTR and P1290S-CFTR (right panel). Login to comment 187 ABCC7 p.Pro439Ser The location of the P439S mutation is also presented on the 3D structures of the MJ0796 dimer (PDBid: 1L2T; the two subunits are represented in green and yellow) and mNBD1 (PDBid: ROZ; orange) superimposed with MJ0796 monomer (PDBid: 1L2TA; green). Login to comment 188 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser Highlights of the possible location of E1401K and P1290S mutations refer to the SAV1866 model. Login to comment 198 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser While P439S and E1401K are close toATP binding sites and therefore likely to affect NBD-dimerization andATP binding, respectively, P1290S is located near the interface NBD/MSD, and may interfere with side-chain contacts within the membrane spanning subunit. Login to comment 200 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser In the present study, we report biochemical and functional data on three CFTR missense mutations located in NBD1 (P439S) and in NBD2 (P1290S, E1401K). Login to comment 210 ABCC7 p.Arg334Trp ABCC7 p.Pro439Ser P439S was identified in heterozygosity with the mild class IV R334W mutation. Login to comment 211 ABCC7 p.Arg334Trp Previous structure and functional studies have shown that R334W is properly processed but produces reduced ion conductance [40]. Login to comment 212 ABCC7 p.Pro1290Ser P1290S was detected in a patient with additional 3272-26A→G mild mutation on the other chromosome. Login to comment 214 ABCC7 p.Glu1401Lys E1401K was identified in compound heterozygosity with the severe F508del mutation. Login to comment 216 ABCC7 p.Arg668Cys ABCC7 p.Pro439Ser Mutation P439S was previously reported in a 10-year old Hispanic boy with the R668C mutation in the other chromosome and with somewhat atypical features of CF such as a sweat Cl- level of 59 mmol/L, mild lung disease and pancreatic insufficiency [45]. Login to comment 217 ABCC7 p.Pro439Ser Based on our present results and since P439S was associated with the very mild R688C mutation [30], it would not be expected a clinical phenotype of pancreatic insufficiency in that patient, as this is usually associated with the presence of severe CFTR mutations [1]. Login to comment 220 ABCC7 p.Pro1290Ser ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser ABCC7 p.Pro439Ser Densitometric analysis showed that the ratio of mature fully-glycosylated to immature core-glycosylated bands detected for P439S-CFTR and P1290S-CFTR was lower than those obtained for wtCFTR, indicating that P439S and P1290S mutations cause dysfunction of CFTR by decreasing total amounts of mature protein. Login to comment 221 ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Sub-cellular localization of the three CFTR mutants in HEK293 cells indicated dramatically reduced membrane staining for P439S-CFTR and P1290S-CFTR and suggests biosynthetic arrest of CFTR maturation similar to F508del-CFTR [43, 47]. Login to comment 222 ABCC7 p.Glu1401Lys In contrast, E1401K-CFTR was correctly localized in the cell membrane, similar to wtCFTR. Login to comment 224 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Thus E1401K probably reduces the activity of CFTR Cl- chan- nels while P439S-CFTR and P1290S-CFTR reduce the number of CFTR channels in the membrane. Login to comment 225 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Further studies using single channel patch clamp will assess if E1401K reduces whole cell conductance by affecting channel open probability or single channel conductance, and if P439S and P1290S also affect CFTR channel pore/gating mechanism. Login to comment 228 ABCC7 p.Pro1290Ser P1290S (proline → serine) is located just before the Q-loop glutamine residue, near the interface between NBD and MSD (Figs. Login to comment 237 ABCC7 p.Glu1401Lys Thus a charge alteration as found in E1401K (glutamic acid → lysine) is likely to disturb ATP binding and therefore reduce CFTR Cl-channel activity, as demonstrated here. Login to comment 240 ABCC7 p.Glu1401Lys Therefore, this suggests that a positive charge introduced by E1401K will reduceATPbinding. Login to comment 247 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Polyphen analysis indicated that while E1401K (PSIC 1.609) is "possibly damaging", P439S (PSIC 2.396) and P1290S (PSIC 2.108) are "probably damaging" (deleterious) to protein function. Login to comment 250 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Although, data obtained with the available bioinformatic tools must be interpreted with caution, these results confirmed experimental data, suggesting that P439S, P1290S and E1401K interfere with protein function. Login to comment 251 ABCC7 p.Glu1401Lys In summary, the combination of reduced CFTR whole-cell conductance and normal protein expression detected for E1401K indicates that this mutation causes a defect in CFTR channel activity probably by reducing ATP binding, as suggested by structural analysis. Login to comment 252 ABCC7 p.Glu1401Lys Thus, E1401K might belong to class III (defective CFTR regulation). Login to comment 253 ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser The other two missense mutations, P439S and P1290S, cause a decrease in the total amounts of the mature protein that might be attributed to a defective processing or an increased protein turnover. Login to comment 254 ABCC7 p.Pro439Ser In fact, P439S seems to be able to impair the fold stability or folding of post-translational NBD1 subunit, which will disrupt the formation of the NBD1/NBD2 heterodimer. Login to comment 255 ABCC7 p.Pro1290Ser Conversely, P1290S appears to affect the NBD1-MSD interaction and thus the overall folding of the entire protein. Login to comment 256 ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Based on present data and in what has been established for the classification of different CFTR mutations, P439S and P1290S might be classified as class V mutations, associated with reduced levels of normally functioning CFTR. Login to comment 258 ABCC7 p.Pro1290Ser ABCC7 p.Pro1290Ser In addition, P1290S is also likely to cause a defective CFTR activity by affecting signalling with the MSD, and therefore P1290S might also be classified as a class III mutation. Login to comment 259 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser This observation might explain the lower whole-cell Cl- currents determined for P1290S-CFTR when compared with P439S-CFTR and E1401K-CFTR. Login to comment 264 ABCC7 p.Glu1401Lys ABCC7 p.Pro1290Ser ABCC7 p.Pro439Ser Although P439S, P1290S and E1401K might not lead to a typical CF phenotpye, the knowledge of the mechanism by which they affect CFTR, improve the ability to interpret and predict the clinical phenotype in patients carrying the studied mutations and may give important insights about the molecular consequences of similar sequence alterations that have been, or remain to be characterized. Login to comment