ABCMdb

PMID: 10585407

Loo TW, Clarke DM
Identification of residues in the drug-binding domain of human P-glycoprotein. Analysis of transmembrane segment 11 by cysteine-scanning mutagenesis and inhibition by dibromobimane.
J Biol Chem. 1999 Dec 10;274(50):35388-92., 1999-12-10 [PubMed]

Sentences

No. Mutations Sentence Comment

69 ABCB1 p.Gly939Val Except for mutant G939V, all of the mutants expressed the 170-kDa protein as the major product. Login to comment 70 ABCB1 p.Gly939Val The major product in mutant G939V was a 150-kDa core-glycosylated protein that was sensitive to endoglycosidase H (data not shown). Login to comment 71 ABCB1 p.Gly939Val In the presence of cyclosporin A, however, all of the mutants, including mutant G939V, yielded the fully mature 170-kDa protein (Fig. 3). Login to comment 72 ABCB1 p.Gly939Val It appeared that mutation G939V affected folding and maturation of P-gp. Login to comment 76 ABCB1 p.Gly939Val ABCB1 p.Tyr953Ala The lowest activity was observed in mutant G939V (62% of wild-type activity), while mutant Y953A had the highest (205%) activity. Login to comment 77 ABCB1 p.Phe957Ala ABCB1 p.Thr945Ala There was moderate stimulation of verapamil-stimulated ATPase activities in mutants T945A (140%), G955V (143%), and F957A (126%). Login to comment 79 ABCB1 p.Tyr953Ala ABCB1 p.Thr945Ala ABCB1 p.Gln946Ala ABCB1 p.Phe942Ala ABCB1 p.Ala947Leu Wild-type P-gp had an apparent affinity of 24 ␮M verapamil, while mutants F942A, T945A, Q946A, A947L, and Y953A had decreased apparent affinities of 93, 100, 165, 156, and 110 ␮M, respectively. Login to comment 80 ABCB1 p.Gly939Val One mutant, G939V, showed the largest increase in apparent affinity for verapamil (8 ␮M). Login to comment 82 ABCB1 p.Thr945Ala One mutant, T945A, showed a pronounced increase in activity (165% of wild-type enzyme). Login to comment 83 ABCB1 p.Phe942Ala ABCB1 p.Ala954Leu By contrast, mutants A954L and F942A had only 13 and 30%, respectively, of the wild-type activity. Login to comment 84 ABCB1 p.Gly939Val ABCB1 p.Tyr953Ala ABCB1 p.Phe957Ala ABCB1 p.Gln946Ala ABCB1 p.Ala947Leu Moderate decreases in activity (40-50%) were observed for mutants G939V, Q946A, A947L, Y953A, and F957A. Login to comment 85 ABCB1 p.Ala947Leu The mutant, A947L, also exhibited a decrease in the apparent affinity for vinblastine. Login to comment 97 ABCB1 p.Ala954Leu The vinblastine-stimulated ATPase activity of mutant A954L was too low for accurate determination of its apparent affinity. Login to comment 99 ABCB1 p.Gly939Val ABCB1 p.Tyr953Ala ABCB1 p.Cys956Ala Its maximal colchicine-stimulated ATPase activity was 220% of that of wild-type P-gp, while those of mutants G939V, C956A, and Y953A were moderately increased (165, 145, and 131%, respectively). Login to comment 100 ABCB1 p.Phe957Ala ABCB1 p.Gln946Ala ABCB1 p.Phe942Ala There were, however, significant decreases in the activity for mutants Q946A (18%), F942A (24%), and F957A (32%). Login to comment 102 ABCB1 p.Gly939Val ABCB1 p.Ala947Leu Wild-type P-gp had an apparent affinity of 620 ␮M colchicine, while those of mutants A947L and G939V were 1870 and 260 ␮M colchicine, respectively. Login to comment 111 ABCB1 p.Phe957Cys All the mutants, except F957C, yielded the mature 170-kDa protein as the major product (data not shown). Login to comment 112 ABCB1 p.Phe957Cys Mutant F957C may be unstable and susceptible to proteolytic digestion. Login to comment 113 ABCB1 p.Phe957Cys Immunoblots of cell extracts of HEK 293 transfected with mutant F957C showed the presence of only degradation products (data not shown). Login to comment 117 ABCB1 p.Gly939Cys Mutant G939C had the lowest activity (70% of that of Cys-less P-gp). Login to comment 120 ABCB1 p.Gly939Cys ABCB1 p.Phe942Cys ABCB1 p.Tyr953Cys ABCB1 p.Thr945Cys Fig. 5 shows that the majority of the mutants, except for G939C, F942C, T945C, and Y953C, were not affected by treatment with dBBn. Login to comment 121 ABCB1 p.Tyr953Cys The activities of mutants F943C and Y953C were slightly inhibited by dBBn (38 and 26% respectively). Login to comment 122 ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys In contrast, the activities of mutants F942C and T945C, were almost completely inhibited by dBBn (80 and 85%, respectively). Login to comment 123 ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys The activities of mutants F942C and T945C were inhibited to FIG. 3. Login to comment 128 ABCB1 p.Thr941Ala ABCB1 p.Phe938Ala ABCB1 p.Gly939Val ABCB1 p.Tyr953Ala ABCB1 p.Phe957Ala ABCB1 p.Thr945Ala ABCB1 p.Gln946Ala ABCB1 p.Phe942Ala ABCB1 p.Ala947Leu ABCB1 p.Ala954Leu ABCB1 p.Cys956Ala ABCB1 p.Ile940Ser ABCB1 p.Phe944Ala ABCB1 p.Phe951Ala ABCB1 p.Ser952Ala ABCB1 p.Met949Ala ABCB1 p.Ile937Ser ABCB1 p.Ser943Ala ABCB1 p.Met948Ala ABCB1 p.Tyr950Ala TABLE I Drug-stimulated ATPase activity Mutant Drug Verapamil Vinblastine Colchicine Vmax Km Vmax Km Vmax Km % of WTa ␮M % of WT ␮M % of WT mM WT 100 24 100 5.4 100 0.62 I937S 94 22 93 6.1 100 0.69 F938A 106 32 96 5.1 96 0.68 G939V 62 8 45 4.0 165 0.26 I940S 93 32 93 5.6 93 0.65 T941A 100 25 104 5.5 100 0.66 F942A 88 93 30 5.1 24 0.80 S943A 92 26 100 5.2 85 0.62 F944A 93 14 105 5.3 101 0.64 T945A 140 100 165 8.3 56 0.65 Q946A 101 165 57 8.5 18 0.64 A947L 105 156 60 13.0 51 1.87 M948A 103 23 101 5.9 103 0.62 M949A 82 40 96 5.5 61 0.60 Y950A 109 37 119 5.1 99 0.62 F951A 94 31 99 5.2 101 0.64 S952A 108 36 123 5.1 91 0.69 Y953A 205 110 59 8.5 131 0.67 A954L 108 44 13 NDb 8 ND G955V 143 10 104 3.5 220 0.47 C956A 97 24 95 5.3 145 0.63 F957A 126 21 47 4.8 32 1.0 a WT, wild type. b ND, not determined due to low activity. Login to comment 138 ABCB1 p.Phe942Cys ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys ABCB1 p.Thr945Cys By contrast, the verapamil-, vinblastine-, or colchicine-stimulated ATPase activities of mutants F942C or T945C were inhibited by more than 70% when pretreated with dBBn. Login to comment 139 ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys We then tested whether the presence of verapamil, vinblastine, or colchicine could protect mutants F942C or T945C from inactivation by dBBn. Login to comment 143 ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys The activities of mutants F942C and T945C, however, were protected by pretreatment with substrate (Fig. 6B). Login to comment 147 ABCB1 p.Gly939Val ABCB1 p.Tyr953Ala ABCB1 p.Thr945Ala ABCB1 p.Gln946Ala ABCB1 p.Phe942Ala ABCB1 p.Ala947Leu DISCUSSION Mutants G939V, F942A, T945A, Q946A, A947L, and Y953A in TM11 had altered apparent affinities for verapamil, vinblastine, or colchicine. Login to comment 148 ABCB1 p.Ala954Leu Mutation of residue Ala954 to leucine appeared to disrupt P-gp-substrate interactions, since little drug-stimulated ATPase activity could be detected in the presence of vinblastine or colchicine. Login to comment 157 ABCB1 p.Thr945Cys Protection of mutant T945C from dBBn inhibition by drug substrates. Login to comment 159 ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys Histidine-tagged Cys-less and mutants F942C and T945C P-gp were transiently expressed in HEK 293 cells in the presence of 10 ␮M cyclosporin A and isolated by nickel-chelate chromatography. Equivalent amounts of each P-gp were mixed with lipid and incubated with 1 mM dBBn for 5 min at 37 °C, quenched with cysteine, and then assayed for verapamil (1 mM)-, vinblastine (0.1 mM)-, or colchicine (5 mM)-stimulated ATPase activity. Login to comment 161 ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys Equivalent amounts of histidine-tagged Cys-less or mutants F942C or T945C P-gp were preincubated for 15 min at 4 °C without or with 2 mM verapamil (Ver. Login to comment 176 ABCB1 p.Gly185Val Mutant G185V is an example of a mutation that probably alters P-gp-drug interactions because of structural perturbations. Login to comment 178 ABCB1 p.Gly185Val Subsequent analysis of this mutant, however, led to the conclusion that G185V alters function of P-gp by affecting the structure of the transporter (49). Login to comment 179 ABCB1 p.Gly939Val ABCB1 p.Ala947Leu ABCB1 p.Ala954Leu Similarly, it is also likely that some of the mutations in TM11 in this study that resulted in altered drug-stimulated ATPase activity could also be due to structural perturbations, since relatively small residues were replaced with larger ones (G939V, A947L, A954L, and G955V). Login to comment 181 ABCB1 p.Gly341Cys An example of an extreme case is mutant G341C (in TM6) that caused complete misfolding of P-gp such that the mutant P-gp was more susceptible to digestion in the first extracellular loop (50). Login to comment 183 ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys Reaction of dBBn with two mutants, F942C and T945C, significantly inhibited substrate-stimulated ATPase activity. Login to comment 184 ABCB1 p.Phe942Cys ABCB1 p.Thr945Cys The presence of verapamil, vinblastine, or colchicine protected mutants F942C and T945C from inhibition by dBBn. Login to comment 194 ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys The helices are also oriented to take into account the cross-linkable nature of residues 346 (TM6) and 989 (TM12) in the mutant G346C/G989C (25). Login to comment