ABCMdb

PMID: 10386624

Fanen P, Clain J, Labarthe R, Hulin P, Girodon E, Pagesy P, Goossens M, Edelman A
Structure-function analysis of a double-mutant cystic fibrosis transmembrane conductance regulator protein occurring in disorders related to cystic fibrosis.
FEBS Lett. 1999 Jun 11;452(3):371-4., [PubMed]

Sentences

No. Mutations Sentence Comment

13 ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp We have now investigated the structure-function relationships of one of these mutants, R74W-D1270N-CFTR, using a heterologous expression system. Login to comment 14 ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp The 'rst mutation lies in the intracellular N-terminus of the CFTR protein and the second within the second nucleotide binding domain (NBD2), replacing an arginine by a tryptophan at position 74 and an aspartic acid by an asparagine at position 1270 [9,10]. Login to comment 20 ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp These assays showed that the R74W mutation should be considered to be a polymorphism and suggest that D1270N alone is su¤cient to produce the CBAVD phenotype, while the combination of the two increases the chloride conductance pathway dysfunction and the phenotype. Login to comment 87 ABCC7 p.Arg74Trp The majority (67%) of the R74W-CFTR cells showed an increased rate of change in MEQ £uorescence when exposed to the stimulatory cocktail, indicating activation of a cAMP-dependent anion pathway, 45% of these cells were fast responders. Login to comment 88 ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Arg74Trp ABCC7 p.Arg74Trp Lastly, 89% of D1270N-CFTR and 81% of R74W-D1270N-CFTR also had a cAMP-responsive anion conductance with di¡erent ratios between fast and slow responding cells (38% fast in D1270N-CFTR and 24% fast in R74W-D1270-CFTR). Login to comment 90 ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Arg74Trp Discussion We have analyzed the structure-function relationships of three CFTR mutations, R74W, D1270N and the R74W-D1270N double-mutant, and compared their properties with those of wild-type CFTR. Login to comment 91 ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp R74W was 'rst described in isolation [10], but has since been found in association with D1270N (Mireille Claustres, personal communication). Login to comment 92 ABCC7 p.Asp1270Asn D1270N was found in a CBAVD patient bearing the vF508 common mutation on the other allele [9]. Login to comment 93 ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp Finally, the R74W-D1270N double-mutant was 'rst identi'ed in a compound, vF508, heterozygote with clinical features of CBAVD, rhinitis, recurrent respiratory infections and elevated sweat chloride concentrations [13]. Login to comment 97 ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp To our knowledge, this is the 'rst reported expression of the R74W- and/or D1270N-CFTR mutant and the 'rst functional description of a double-mutant associated with CBAVD, a disorder related to CF. Login to comment 104 ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Arg74Trp HeLa cells were transfected with either wild-type CFTR (vFstim/vFbasal = 12), R74W-CFTR (vFstim/vFbasal = 11), D1270N-CFTR (vFstim/vFbasal = 3) or R74W-D1270N-CFTR (vFstim/ vFbasal = 2). Login to comment 105 ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Arg74Trp Table 1 Summary of the MEQ assay results Cell type Wild-type R74W D1270N R74W-D1270N PTracer Total 53 30 27 26 28 Non-responding 8 10 3 5 28 All responding 45 (85%)a 20 (67%)a 24 (89%)a 21 (81%)a 0 Fast 21 (47%)b 9 (45%)b 9 (38%)b 5 (24%)b 0 vFstim/vFbasal 13.3 þ 6.3c 11.9 þ 8.7c 7.7 þ 2.4c 7.3 þ 2.7c Range 5.4^26.3 5.9^34 5.6^12.8 5.2^12 Slow 24 (53%)b 11 (55%)b 15 (62%)b 16 (76%)b 0 vFstim/vFbasal 2.7 þ 1.1c 2.5 þ 0.9c 2.8 þ 0.9c 3.2 þ 1.2c Range 1.3^5 1.1^4.1 1.4^4.5 1.2^4.9 a Percentage of all cells. Login to comment 107 ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Arg74Trp c Mean values þ S.D. FEBS 22106 -6-99 P. Fanen et al./FEBS Letters 452 (1999) 371^374 373 Expression of these three mutant genes in HeLa cells showed that R74W-CFTR, D1270N-CFTR and R74W-D1270N-CFTR proteins elicited cAMP-dependent chloride £uxes. Login to comment 108 ABCC7 p.Arg74Trp R74W-CFTR elicited a cAMP-responsive anion conductance at the same rate as wild-type CFTR. Login to comment 110 ABCC7 p.Arg74Trp On the basis of the results of the functional assay, we postulate that the R74W mutation is a sequence variation that has no deleterious e¡ect alone. Login to comment 111 ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp D1270N and R74W-D1270N had abnormal responsive patterns. Login to comment 113 ABCC7 p.Arg117His ABCC7 p.Asp1270Asn This fraction, as well as that of D1270N-CFTR (38%), resembles the pattern of responses of R117H-CFTR, another mild mutant associated with CBAVD [11]. Login to comment 115 ABCC7 p.Arg117His The R117H mutation produces di¡erent phenotypes, depending on the presence or absence of the 5T allele on the same chromosome [17]. Login to comment 116 ABCC7 p.Arg117His When R117H occurs on a 5T background, the physiology of the vas deferens, lungs and sweat glands is impaired, de'ning a complete but mild CF. Login to comment 117 ABCC7 p.Arg117His When R117H occurs on a 7T background, only the physiology of the vas deferens is impaired. Login to comment 118 ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp The R74W-D1270N double-mutant was associated with a 7T background in the three patients studied. Login to comment 121 ABCC7 p.Arg117His Thus, this double-mutant strongly resembles the R117H mutation when it is associated with the 7T background. Login to comment 122 ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp We therefore believe that the R74W-D1270N double-mutant is responsible for the CBAVD phenotype. Login to comment 123 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp The contribution of each mutant to this phenotype may be as follows: R74W is a polymorphism which may slightly reduce the normal amount of CFTR protein (67% of responding cells versus 81^89%) in vivo and D1270N has a cAMP-responsive anion conductance with di¡erent ratios between fast and slow responder cells, as for the R117H mutation. Login to comment 124 ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp ABCC7 p.Arg74Trp ABCC7 p.Arg74Trp We infer that the combination of R74W enhances the e¡ect of D1270N by reducing the number of fast responder cells, as a consequence of the defective regulation of the R74W-D1270N mutated protein or of a di¡erent turn-over of the protein at the cell surface in vivo. Login to comment 125 ABCC7 p.Asp1270Asn ABCC7 p.Asp1270Asn ABCC7 p.Arg74Trp This is supported by the fact that the compound heterozygote vF508/ D1270N described by Anguiano et al. occurred in a CBAVD patient with normal sweat sodium and chloride values, i.e. normal chloride secretion [9], whereas the compound heterozygote vF508/R74W-D1270N described by Casals et al. had a more severe phenotype and elevated sweat chloride concentrations suggesting an abnormal chloride secretion [13]. Login to comment