ABCMdb

ABCB1 p.Asn296Ala

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Publications

PMID: 21182301 [PubMed] Loo TW et al: "The W232R suppressor mutation promotes maturation of a truncation mutant lacking both nucleotide-binding domains and restores interdomain assembly and activity of P-glycoprotein processing mutants."

No. Sentence Comment

84 Baby hamster kidney (BHK) cells expressing A52-tagged wild-type P-gp or mutants G251V, G251V/W232R, W232R, W232A, N296A, E875A, or T945A were generated as described previously (25). Login to comment
167 The N296A Mutation Abolishes W232R Rescue. Login to comment
184 It was observed that only one mutation in TM5 (N296A) inhibited maturation of the G251V/W232R mutant when it was expressed in the absence of cyclosporin A (Figure 4C, lane 7). Login to comment
186 Introduction of the N296A mutation into G251V/W232R(TM4) caused the mutant to behave in a fashion similar to the original FIGURE 3: Effect of W232R on cross-linking between domains of processing mutants. Login to comment
194 (C) HEK 293 cells transfected with A52-tagged mutant G251V or G251V/ W232R containing alanine mutations in potential hydrogen-bonding residues in TM5 (T294A, N296A, S298A) or G251V/N296A were expressed in the absence (-) or presence (þ) of cyclosporin A (cyclo A). Login to comment
200 Like the G251V parent (Figure 4C, lanes 1 and 2), the G251V/W232R/N296A mutant (Figure 4C, lane 7) showed about 15% maturation efficiency when expressed in the absence of drug substrates, and the mature 170 kDa P-gp was the major product when expressed in the presence of cyclosporin A (Figure 4C, lane 8). Login to comment
201 Therefore, the presence of the N296A mutation prevented the ability of W232R to promote maturation of the G251V mutant. Login to comment
202 Figure 4A (lanes 11 and 12) shows that the N296A change alone had little effect on the maturation characteristics of the G251V parent because it could still be rescued with cyclosporin A. Login to comment
203 These results suggest that Arg232(TM4) promotes maturation of the G251V mutant through hydrogen bond interactions with Asn296(TM5) because the N296A mutation inhibited the ability of W232R to promote maturation of G251V (Figure 4C, lane 7). Login to comment
219 No detectable mature protein was observed in cells expressing TMD1 þ 2 lacking W232R or with mutant TMD1 þ 2(W232R/N296A) (Figure 6A). Login to comment
229 (A) Whole cell extracts of cells transfected with A52-tagged wild-type, W232R, or W232R/N296A forms of TMD1 þ 2 were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment
285 Stable BHK cell lines expressing mutants W232A, N296A, E875A, or T945A were generated. Login to comment
289 For example, cells expressing mutants W232A and N296A were more resistant to colchicine (P < 0.05) than cells expressing wild-type P-gp, but their resistance to paclitaxel was reduced (P < 0.05). Login to comment
299 Each value is average of triplicate assays ( SD. (B) Whole cell extracts of BHK cells expressing vector (control), A52-tagged wild-type, mutant W232A, N296A, E875A, or T945A P-gp were subjected to immunoblot analysis with monoclonal antibody against A52 or GAPDH. Login to comment
304 The verapamil-stimulated ATPase activities of mutants W232A and N296A, however, resembled that of wild-type enzyme (S50 of 30 and 42 μM, respectively). Login to comment
306 The maximum activity was increased by 50% in mutant N296A when compared to wild-type P-gp. Login to comment
308 The S50 of mutants W232A (45 μM) and T945A (200 μM) differed from wild-type P-gp (102 μM) while those of mutants N296A (78 μM) and E875A (86 μM) were similar. Login to comment
310 In summary, the E875A and T945A mutations altered both verapamiland rhodamine B-stimulated ATPase activity while the W232A and N296A mutations only affected rhodamine B-stimulated ATPase activity. Login to comment
336 Histidine-tagged wild-type or mutant W232A, N296A, E875A, or T945A P-gp was expressed in HEK 293 cells and isolated by nickel-chelate chromatography. Login to comment