ABCMdb

PMID: 17244607

Zegarra-Moran O, Monteverde M, Galietta LJ, Moran O
Functional analysis of mutations in the putative binding site for cystic fibrosis transmembrane conductance regulator potentiators. Interaction between activation and inhibition.
J Biol Chem. 2007 Mar 23;282(12):9098-104. Epub 2007 Jan 23., 2007-03-23 [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Arg553Gln We found that all three mutants responded to cAMP, although the affinity of R553Q was higher than that of wild type CFTR. Login to comment 6 ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly In R553Q and V1293G mutants, the dissociation constant of potentiators for the activating site was increased, whereas the dissociation constant for the inhibitory site was reduced. Login to comment 37 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Printed in the U.S.A. 9098 tion that mutations in conserved residues of the NBDs such as G551D and G1349D exhibit a shift in the affinity for potentiators (5, 15-18). Login to comment 41 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp After in silico docking of several compounds, we compared the theoretical binding free energy measured on the model, with the experimental binding free energy obtained from dissociation constants from wild type, G551D, and G1349D proteins. We found a good correlation between these two parameters for a putative binding site located in the interface of the NBD1-NBD2 dimer, embedded in a cavity on NBD1, and interacting also with the NBD2 surface. Login to comment 44 ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly We found that the stimulating effect of potentiators was reduced for R553Q and V1293G. Login to comment 49 ABCC7 p.Val1293Gly Mutant V1293G was introduced in a wild type CFTR construct contained in the expression vector pTracer-CMV (15) by a recombinant PCR method. Login to comment 50 ABCC7 p.Arg553Gln ABCC7 p.Ala554Glu For mutations A554E and R553Q, CFTR cDNA was subcloned on pcDNA3.1. Login to comment 52 ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu V1293G clones were selected and maintained in 800 ␮g/ml Zeocin, and A554E and R553Q constructs were selected and maintained in 1 mg/ml G418. Login to comment 53 ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu Cell Cultures-FRT cells expressing wild type (WT), V1293G, A554E, or R553Q CFTR were cultured on 60-mm Petri dishes with Coon`s modified F12 containing 5% fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 ␮g/ml streptomycin and selection antibiotics, as described previously (15). Login to comment 85 ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu The apparent dissociation constant of CPTcAMP for mutants V1293G and A554E was not statistically different from that of the wild type protein (Fig. 2). Login to comment 86 ABCC7 p.Arg553Gln Conversely, the dissociation constant for R553Q was significantly smaller. Login to comment 98 ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu Continuous lines (WT, A554E, and V1293G) and broken lines (R553Q) indicate fitting of the data to Equation 1. Login to comment 99 ABCC7 p.Gly551Asp TABLE 1 Parameters obtained from the fit to Equation 1 of CPTcAMP dose-response relationship on wild type and mutant CFTRs For comparison, the parameters of G551D, a severe CF-causing mutation, are included. Login to comment 100 ABCC7 p.Gly551Asp ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu Wild type A554E V1293G R553Q G551D n ϭ 5 n ϭ 4 n ϭ 4 n ϭ 6 n ϭ 7 Imax (␮A/cm2 ) 282.2 Ϯ 13.5 66.9 Ϯ 16.4a 70.2 Ϯ 14a 93.8 Ϯ 19a 10.1 Ϯ 2.8a Kd (␮M) 54.2 Ϯ 11.5 40.5 Ϯ 5.9 48.4 Ϯ 13.5 21.5 Ϯ 4.5a 74.2 Ϯ 12.1 I(20)/I(max) 0.3 Ϯ 0.04 0.34 Ϯ 0.03 0.32 Ϯ 0.05 0.51 Ϯ 0.05a 0.23 Ϯ 0.03 a p Ͻ 0.05. Login to comment 102 ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu It is interesting to note that 20 ␮M CPTcAMP produced a similar current fraction (ϳ0.3) on mutants V1293G and A554E but half of the maximum current (ϳ0.5) on R553Q (see I(20)/ I(max) in Table 1), in agreement with the lower Kd of CPT-cAMP found on this mutant. Login to comment 107 ABCC7 p.Ala554Glu The response of mutant A554E to genistein, with almost no change on Ka or Ki, was similar to the response of wild type CFTR. Login to comment 108 ABCC7 p.Arg553Gln In contrast, the protein with the adjacent residue mutated, R553Q, behaved in a completely different way. Login to comment 110 ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu Mutant V1293G behavior was between A554E and R553Q. Login to comment 111 ABCC7 p.Arg553Gln In fact, the two equilibrium constants were modified but less than in mutant R553Q (Table 2). Login to comment 113 ABCC7 p.Val1293Gly In general, for these compounds Ka changed on mutants in the same direction as Ka for genistein; however, the effect was less marked, except for mutant V1293G, which tended to be more sensitive to Act-06 (see Table 2). Login to comment 120 ABCC7 p.Arg553Gln In fact, the more the Ka value shifted toward higher concentrations, as in the case of genistein for R553Q, the more the Ki value shifted toward lower concentrations. Login to comment 123 ABCC7 p.Arg553Gln A, representative traces showing the response of wild type CFTR and mutant R553Q to application of genistein. Login to comment 127 ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu C-E, comparison of normalized and averaged genistein dose-response relationships of mutants V1293G,A554E,andR553Q(seesymbolkeys)toWT(dashedlines).Eachsymbol is the mean of 4-6 experiments, and vertical bars show S.E. Login to comment 130 ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly ABCC7 p.Val1293Gly ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu ABCC7 p.Ala554Glu ABCC7 p.Ala554Glu Compounds Protein n fA Ka Ki ␮M ␮M Genistein Wild type 5 1.33 Ϯ 0.41 3.08 Ϯ 0.74 562.4 Ϯ 111.1 A554E 6 2.75 Ϯ 0.62 4.58 Ϯ 0.51 408.6 Ϯ 84.8 V1293G 6 4.87 Ϯ 1.91 12 Ϯ 3.9a 270.5 Ϯ 36.4a R553Q 6 1.87 Ϯ 0.12 22.28 Ϯ 5.2a 119.9 Ϯ 34.9a UCCF029 Wild type 4 0.41 Ϯ 0.04 0.021 Ϯ 0.002 1036 Ϯ 523 A554E 4 1.56 Ϯ 0.45a 0.036 Ϯ 0.009 1519 Ϯ 651 V1293G 5 3.19 Ϯ 1.09 0.042 Ϯ 0.008 843 Ϯ 126 R553Q 5 1.08 Ϯ 0.17a 0.154 Ϯ 0.029a 547 Ϯ 99 Act-06 Wild type 5 0.8 Ϯ 0.17 0.69 Ϯ 0.33 439.6 Ϯ 108 A554E 4 1.67 Ϯ 0.42 0.74 Ϯ 0.25 319.1 Ϯ 42.4 V1293G 4 1.25 Ϯ 0.16 0.35 Ϯ 0.09 383.8 Ϯ 28.9 R553Q 6 1.77 Ϯ 0.47a 2.11 Ϯ 0.73 179.1 Ϯ 73.2a a Student`s t test indicated that these values were statistically different from those on WT CFTR with p Ͻ 0.05. Login to comment 149 ABCC7 p.Arg553Gln Surprisingly, we found a lower equilibrium constant for CPT-cAMP on mutant R553Q (see Table 2). Login to comment 150 ABCC7 p.Gly551Asp ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu Mutations of NBDs, like the CF mutation G551D (see Table 1 and Ref. 15) or the other mutations studied here, A554E and V1293G, do not seem to change significantly the sensitivity of the protein to CPTcAMP. Login to comment 152 ABCC7 p.Arg553Gln It is interesting to note that mutation R553Q is one of the CF mutations identified as ⌬Phe508 revertants (34). Login to comment 153 ABCC7 p.Arg553Gln That means that introduction of R553Q mutation into human CFTR partially corrects the processing and gating defect caused by the ⌬Phe508 mutation, which has been suggested to modify the NBD1 surface that interacts with other CFTR domains (20). Login to comment 155 ABCC7 p.Gly551Asp An even more marked reduction had been found previously for G551D (see Table 1 and Refs. Login to comment 156 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp 15 and 18), but in that case we measured the expression of both G551D and wild type proteins. We concluded that the small amount of G551D current was due to a severe gating defect of the mutant and not to reduced expression levels, as confirmed by the reduced open channel probability estimated from single channel recording. Login to comment 159 ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu Conversion of the Ala554 residue to glutamic acid had little effect on the capability of potentiators to favor the conductive state, whereas conversion of Val1293 to glycine increased the Ka for genistein by 4-fold. Login to comment 163 ABCC7 p.Arg553Gln In fact, the mutation that produced a stronger shift of the affinity for these compounds was R553Q, where the charge was eliminated. Login to comment 167 ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly ABCC7 p.Ala554Glu The CFTR proteins are indicated in red, yellow, green, and blue for R553Q, V1293G, A554E and WT, respectively. Login to comment 169 ABCC7 p.Ala554Glu Mutations in Binding Site for CFTR Potentiators 9102 potentiators (see Fig. 1; the distance between these amino acids and potentiators is less than 3.6 Å, whereas in A554E this distance is larger), and therefore a modification of any of them is predicted to produce a strong effect on the activation dissociation constant. Login to comment 170 ABCC7 p.Ala554Glu The A554E mutant is different. Login to comment 176 ABCC7 p.Arg553Gln ABCC7 p.Val1293Gly Actually, we found that an increased Ka in R553Q and V1293G with respect to wild type CFTR is accompanied by a reduced Ki (Figs. Login to comment 186 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp Probably, the NBD with higher affinity for potentiators is NBD1, because CF mutation G551D, situated on NBD1 near the potentiator binding site, causes a more pronounced effect on the equilibrium constant for the activation site than the symmetrical CF mutation on NBD2, G1349D (16). Login to comment