ABCMdb

PMID: 9405384

Loo TW, Clarke DM
Identification of residues in the drug-binding site of human P-glycoprotein using a thiol-reactive substrate.
J Biol Chem. 1997 Dec 19;272(51):31945-8., 1997-12-19 [PubMed]

Sentences

No. Mutations Sentence Comment

21 ABCB1 p.Leu975Cys ABCB1 p.Leu339Cys ABCB1 p.Ala342Cys ABCB1 p.Val982Cys ABCB1 p.Ala985Cys We show that the drug-stimulated ATPase activities of mutants L339C and A342C (TM6) and L975C, V982C, and A985C (TM12) were particularly sensitive to inhibition by dBBn and that the inhibition was prevented by various drug substrates. Login to comment 62 ABCB1 p.Ser344Cys The activity of mutant S344C was not determined (ND) due to our inability to express enough enzyme. Login to comment 65 ABCB1 p.Asn91Ala ABCB1 p.Asn94Ala ABCB1 p.Asn99Ala B, whole cell extracts of HEK 293 cells expressing wild-type, Cys-less, glycosylation-deficient N91A/N94A/N99A and single Cys mutants that exhibited little or no verapamil-stimulated ATPase activity were subjected to immunoblot analysis as described under "Experimental Procedures." Login to comment 67 ABCB1 p.Ser344Cys For mutant S344C, three times the normal amount of lysate was loaded onto the gel. Login to comment 81 ABCB1 p.Phe335Cys ABCB1 p.Phe978Cys One mutant, F335C (TM6) showed enhanced activity (280%), whereas the equivalent residue in TM12 (F978C) showed decreased activity (31%). Login to comment 83 ABCB1 p.Gly341Cys ABCB1 p.Gln347Cys ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Ala342Cys ABCB1 p.Gly984Cys ABCB1 p.Ala985Cys ABCB1 p.Ser344Cys ABCB1 p.Gln990Cys There was no detectable activity with mutants S344C, G341C, and G984C, whereas mutants A342C, G346C, Q347C, A985C, G989C, and Q990C had much reduced activity (10-40%). Login to comment 86 ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Ala985Cys ABCB1 p.Gln990Cys A similar pattern was observed for mutants G346C, A985C, G989C, and Q990C, suggesting that the low ATPase activity in these mutants was not due to a processing defect. Login to comment 87 ABCB1 p.Gly341Cys ABCB1 p.Gly984Cys Mutants G341C and G984C, however, appeared to be degraded quite rapidly. Login to comment 89 ABCB1 p.Gln347Cys ABCB1 p.Ala342Cys Mutants A342C and Q347C also showed enhanced degradation in the absence of cyclosporin A, with the 120-kDa protein as the major product. Login to comment 90 ABCB1 p.Asn91Ala ABCB1 p.Asn94Ala ABCB1 p.Asn99Ala This appeared to be a degradation product rather than a nonglycosylated product because it had a higher mobility than the glycosylation-deficient P-glycoprotein (N91A/N94A/N99A) (Fig. 2B, lanes 23 and 24). Login to comment 92 ABCB1 p.Ser344Cys Mutant S344C consistently yielded very low levels of immunoreactive P-glycoprotein in the presence or the absence of cyclosporin A (Fig. 2B, lanes 13 and 14). Login to comment 96 ABCB1 p.Gly341Cys ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Gly984Cys ABCB1 p.Ser344Cys Mutants G341C, S344C, G346C, G984C, and G989C were not assayed because of their low or defective expression (Fig. 2B). Login to comment 98 ABCB1 p.Leu975Cys ABCB1 p.Leu339Cys ABCB1 p.Ala342Cys ABCB1 p.Val982Cys ABCB1 p.Ala985Cys In contrast, mutants L339C, A342C, L975C, V982C, and A985C were significantly inhibited by dBBn, because they retained only 10, 40, 13, 25, and 32% of their activities, respectively. Login to comment 99 ABCB1 p.Leu975Cys ABCB1 p.Leu339Cys ABCB1 p.Ala342Cys ABCB1 p.Val982Cys ABCB1 p.Ala985Cys The concentration of dBBn required to give 50% inhibition of ATPase activity for mutants L339C, L975C, V982C, A985C, and A342C were 90, 112, 320, 480, and 700 ␮M, respectively. Login to comment 106 ABCB1 p.Ser344Cys Inhibition of the activity of mutant S344C was not determined (ND) due to low expression, and mutants indicated by asterisks were not assayed due to proteolytic digestion. Login to comment 107 ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys The inhibition of mutants G346C and G989C were not determined (ND) due to their low activities. Login to comment 111 ABCB1 p.Leu975Cys ABCB1 p.Leu339Cys ABCB1 p.Ala342Cys ABCB1 p.Val982Cys ABCB1 p.Ala985Cys The P-glycoproteins(His)10 of Cys-less and mutants L339C, A342C, L975C, V982C, and A985C were mixed with lipid and then preincubated for 15 min at 4 °C without drug or in the presence of 2 mM verapamil (Ver. Login to comment 114 ABCB1 p.Ala342Cys ABCB1 p.Ala985Cys Mutants A342C and A985C were preincubated with verapamil only. Login to comment 121 ABCB1 p.Ala342Cys ABCB1 p.Ala985Cys Due to the low ATPase activities of mutants A342C and A985C, their protection assays were done only in the presence of verapamil. Login to comment 123 ABCB1 p.Ala342Cys ABCB1 p.Ala985Cys As shown in Fig. 4, mutants A342C and A985C were protected from dBBn inactivation by verapamil. Login to comment 124 ABCB1 p.Leu975Cys ABCB1 p.Leu339Cys ABCB1 p.Val982Cys Similarly, mutants L339C, L975C, and V982C were also protected from dBBn inactivation by various drug substrates. Login to comment 126 ABCB1 p.Leu339Cys Colchicine was also very effective in protecting mutant L339C from dBBn inactivation because it retained about 80% of its colchicine-stimulated ATPase activity. Login to comment 127 ABCB1 p.Leu975Cys ABCB1 p.Val982Cys More modest protection by colchicine was seen for mutants L975C and V982C. Login to comment 129 ABCB1 p.Leu975Cys ABCB1 p.Leu339Cys ABCB1 p.Val982Cys It offered little or no protection for mutant V982C and only moderately protected mutants L339C and L975C. Login to comment 141 ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Phe343Cys ABCB1 p.Met986Cys Cross-linking between residues F343C/M986C, G346C/G989C, and P350C/S993C was prevented by the presence of drug substrates. Login to comment