ABCMdb

PMID: 11118444

Clain J, Fritsch J, Lehmann-Che J, Bali M, Arous N, Goossens M, Edelman A, Fanen P
Two mild cystic fibrosis-associated mutations result in severe cystic fibrosis when combined in cis and reveal a residue important for cystic fibrosis transmembrane conductance regulator processing and function.
J Biol Chem. 2001 Mar 23;276(12):9045-9. Epub 2000 Dec 15., 2001-03-23 [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Arg347His ABCC7 p.Asp979Ala We investigated the structure-function relationships of a severe cystic fibrosis (CF)-associated double mutant (R347H-D979A) to evaluate the contribution of each mild mutation to the phenotype. Login to comment 3 ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala Our data show that R347H is associated with mild defective Cl-channel activity and that the D979A defect leads to misprocessing. Login to comment 4 ABCC7 p.Arg347His ABCC7 p.Asp979Ala The mutant R347H-D979A combines both defects for a dramatic decrease in Cl- cur- rent. Login to comment 13 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala The recent discovery of severe CF associated with a ⌬F508/ R347H-D979A compound heterozygote genotype in two related patients suffering from pancreatic insufficiency and severe respiratory symptoms suggests that the R347H-D979A mutation has an important influence on CFTR processing and/or function (7). Login to comment 14 ABCC7 p.Arg347Pro ABCC7 p.Arg347Cys ABCC7 p.Arg347His ABCC7 p.Arg347Leu ABCC7 p.Asp979Ala ABCC7 p.Asp979Val At least four CF-associated mutations have been identified in isolation at position 347 (R347C, R347H, R347L, and R347P) and two at position 979 (D979A and D979V), suggesting that Arg-347 and Asp-979 are important for CFTR structure and/or function. Login to comment 15 ABCC7 p.Arg347His ABCC7 p.Asp979Ala The mutation D979A was found in isolation in a patient with a congenital bilateral absence of the vas deferens (8) and the R347H mutation in CF patients with pancreatic sufficiency, congenital bilateral absence of the vas deferens, and no or mild pulmonary symptoms (7). Login to comment 16 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp979Ala As the R347H mutation is mostly associated with mild CF, it was suggested that the D979A mutation has a significant effect on CFTR function when combined in cis with R347H. Login to comment 18 ABCC7 p.Arg347His ABCC7 p.Asp979Ala The mutations R347H and D979A replace positively charged (Arg) and negatively charged (Asp) residues with ones that are uncharged (His and Ala, respectively) at physiological pH. Login to comment 19 ABCC7 p.Arg347His ABCC7 p.Asp979Ala The present study investigates the structure-function relationships of the R347H-D979A double mutant, and as charged residues are involved in this complex genotype, single and double mutants with different charge combinations at residues 347 and 979 were constructed. Login to comment 21 ABCC7 p.Arg347His ABCC7 p.Asp979Ala Our data show that R347H is associated with defective chloride channel activity and that the D979A defect leads to misprocessing. Login to comment 22 ABCC7 p.Arg347His ABCC7 p.Asp979Ala The mutant R347H-D979A combines both defects for a dramatic decrease in Cl- current. Login to comment 58 ABCC7 p.Arg347His ABCC7 p.Asp979Ala We first studied the maturation of CFTR in HeLa cells to determine why patients with the R347H-D979A-CFTR allele suffered from severe CF. Login to comment 64 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala Immunoprecipitation experiments show that both wild-type and CF-associated mutants R347H, D979A, and R347H-D979A-CFTR cells produced mature, fully glycosylated protein (Fig. 1A; band C), whereas none of the mock-transfected cells produced CFTR (data not shown). Login to comment 65 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala However, the D979A and R347H-D979A mutants produced significantly less band C (59 and 56% of the total CFTR; ratio C/(BϩC)) than wild-type and R347H-CFTR (92 and 91%; see Fig. 1B). Login to comment 66 ABCC7 p.Arg347His ABCC7 p.Asp979Ala This indicates that the R347H-D979A mutation caused a misprocessing defect. Login to comment 67 ABCC7 p.Arg347His ABCC7 p.Asp979Ala The turnover of immature and mature forms of wild-type and R347H-D979A-CFTR proteins was further investigated by pulse-chase experiments (Fig. 1C). Login to comment 68 ABCC7 p.Arg347His ABCC7 p.Asp979Ala The kinetics of wild-type and R347H-D979A core-glycosylated and mature forms of CFTR were identical, whereas the efficiency of conversion to mature band C was lower for the mutant. Login to comment 69 ABCC7 p.Asp979Ala Similar results were obtained with the D979A mutant (data not shown). Login to comment 71 ABCC7 p.Asp979Ala Other mutants were generated to further characterize the D979A defect. Login to comment 72 ABCC7 p.Asp979Val ABCC7 p.Asp979Val ABCC7 p.Asp979Arg ABCC7 p.Asp979Glu Asp-979 was changed to Val (small hydrophobic residue; D979V), Arg (positively charged residue; D979R), or Glu (negatively charged residue; D979E). Login to comment 73 ABCC7 p.Asp979Val ABCC7 p.Asp979Arg D979V (also a naturally occurring mutant) and D979R had impaired processing FIG. 1. Login to comment 82 ABCC7 p.Arg347His ABCC7 p.Asp979Ala C, pulse-chase experiments showing the turnover of the immature and mature forms of wild-type and R347H-D979A-CFTR (results are representative of two independent experiments). Login to comment 83 ABCC7 p.Asp979Ala ABCC7 p.Asp979Glu similar to D979A, whereas D979E permitted the complete maturation of the protein (Fig. 1B). Login to comment 84 ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala Altogether, these results indicate that D979A is responsible for the defective processing of R347H-D979A-CFTR and that a negative charge at 979 residue is necessary for proper CFTR processing. Login to comment 93 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala We separated the contributions of the R347H and D979A mutations to R347H-D979A-CFTR whole-cell Cl- current production studying both single and double mutants. Login to comment 96 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala Mean changes in cAMP-activated currents recorded at 20 mV from R347H (29.4 Ϯ 12.2 pA/pF; n ϭ 8), D979A (67.9 Ϯ 18.9 pA/pF; n ϭ 5), and R347H-D979A (1.2 Ϯ 0.8 pA/pF; n ϭ 9) mutants were significantly different (p Ͻ 0.05) from wild-type (130.2 Ϯ 34.7 pA/pF; n ϭ 5), corresponding to 23, 52, and 1% of the wild-type Cl- current (Fig. 2D). Login to comment 97 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala R347H, D979A, and R347H-D979A were also significantly different from each other (p Ͻ 0.01). Login to comment 98 ABCC7 p.Arg347His ABCC7 p.Arg347His As R347H processing is similar to wild-type, the small Cl- current produced by R347H reflected defective channel properties, as demonstrated by single-channel studies (9). Login to comment 99 ABCC7 p.Asp979Ala The decreased whole-cell Cl- cAMP-dependent current observed with D979A probably reflected CFTR protein misprocessing, although we cannot firmly exclude altered channel properties. Login to comment 100 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala Thus these data indicate that the R347H-D979A double mutant combined at least D979A misprocessing and the R347H Cl-channel defect to produce a very severe phenotype. Login to comment 101 ABCC7 p.Arg347His ABCC7 p.Arg347Asp ABCC7 p.Asp979Ala ABCC7 p.Asp979Arg Charge-reversal Mutants-Taking into account the functional defects that result when Arg-347 and Asp-979 are each replaced with an uncharged amino acid such as His (uncharged at pH 7.3) and Ala (R347H and D979A), we constructed additional mutants with different charge combinations at residues 347 and 979, including the R347D-D979R double mutant in which the positive and negative charges were swapped. Login to comment 102 ABCC7 p.Arg347His ABCC7 p.Arg347Asp The processing of R347D was similar to those of R347H and the wild-type (Fig. 3, open bars). Login to comment 109 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala C, current-voltage (I-V) curve in HeLa cells expressing R347H (filled circle), D979A (filled triangle), and R347H-D979A (filled square) mutants. Login to comment 118 ABCC7 p.Arg347His ABCC7 p.Arg347Asp ABCC7 p.Asp979Arg ABCC7 p.Asp979Arg ABCC7 p.Asp979Arg essing of D979R, R347H-D979R, and R347D-D979R was differently impaired (Fig. 3; gray bars). Login to comment 120 ABCC7 p.Asp979Arg This suggests that 347 residue could interact directly or indirectly with Arg-979 (D979R). Login to comment 121 ABCC7 p.Asp979Ala ABCC7 p.Asp979Arg Whole-cell measurements indicated that D979R resulted in a much greater decrease in chloride current (4.5 Ϯ 2.4 pA/pF; n ϭ 6) than D979A (Fig. 2D), although both proteins were processed similarly. Login to comment 123 ABCC7 p.Arg347His ABCC7 p.Arg347Asp ABCC7 p.Asp979Ala ABCC7 p.Asp979Arg ABCC7 p.Asp979Arg The Cl- current of R347D-D979R (2.8 Ϯ 1.7 pA/pF; n ϭ 9) was not significantly different from those of D979R and R347H-D979A (Fig. 2D). Login to comment 124 ABCC7 p.Arg347Asp ABCC7 p.Asp979Arg This result is consistent with the poor amount of R347D-D979R protein at the cell surface. Login to comment 125 ABCC7 p.Arg334Trp ABCC7 p.Arg1158* ABCC7 p.Arg553Gln ABCC7 p.Val1212Ile DISCUSSION Complex alleles have been described clinically (R553Q- ⌬F508, ⌬F508-V1212I, and R334W-R1158X), and most of them are considered to reverse the phenotype, as they are associated with milder symptoms than the most common mutation in isolation. Login to comment 127 ABCC7 p.Arg347His ABCC7 p.Asp979Ala Clinical data suggested that R347H and D979A, two mild CF-associated mutations, can produce severe CF similar to that of ⌬F508 homozygotes when combined in cis. Login to comment 129 ABCC7 p.Arg347His ABCC7 p.Asp979Ala D979A reduces the amount of CFTR protein at the cell membrane, whereas R347H generates a defective Cl-channel. Login to comment 130 ABCC7 p.Arg347His ABCC7 p.Asp979Ala The mutant R347H-D979A combines both defects for a dramatic decrease in Cl- current. Login to comment 131 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala The magnitude of Cl- current in vitro paralleled the severity of the disease, with D979A (congenital bilateral absence of the vas deferens) Ͼ R347H (mild CF) Ͼ R347H-D979A (severe CF). Login to comment 134 ABCC7 p.Asp979Arg Our data strongly suggest that mutation D979R alters the properties of the chloride channel, and mutational analysis of 979 residue also confirmed that the requirements for channel processing and function are different, consistent with data for other residues (17, 18, 21). Login to comment 136 ABCC7 p.Asp979Ala ABCC7 p.Asp979Val Our data support this view, as replacing Asp-979 by Ala, Val, or Arg significantly impaired processing. Login to comment 143 ABCC7 p.Arg347His ABCC7 p.Arg347His ABCC7 p.Arg347Asp ABCC7 p.Arg347Asp ABCC7 p.Asp979Ala ABCC7 p.Asp979Ala ABCC7 p.Asp979Val ABCC7 p.Asp979Arg ABCC7 p.Asp979Arg First, several lines of experimental evidence indicate that there is probably no direct salt bridge between Arg-347 and Asp-979: (i) removal of either the positive charge at position 347 (R347H and R347D) or the negative charge at position 979 (D979A, D979V, and D979R) has different effects on CFTR processing; (ii) the double-neutral (R347H-D979A) and reversed-charged (R347D-D979R) replacements for Arg-347 and Asp-979 do not lead to the recovery of wild-type processing. Login to comment 146 ABCC7 p.Asp979Arg This is supported by the decrease in the processing efficiency of D979R mutants combined with positive, neutral, and negative charges at residue 347. Login to comment