ABCMdb

PMID: 9261097

Loo TW, Clarke DM
Drug-stimulated ATPase activity of human P-glycoprotein requires movement between transmembrane segments 6 and 12.
J Biol Chem. 1997 Aug 22;272(34):20986-9., 1997-08-22 [PubMed]

Sentences

No. Mutations Sentence Comment

68 ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Leu339Cys ABCB1 p.Phe343Cys ABCB1 p.Val982Cys ABCB1 p.Met986Cys ABCB1 p.Phe336Cys ABCB1 p.Ser979Cys To test these predictions, we introduced pairs of cysteines into a Cys-less mutant of P-glycoprotein to create the mutants F336C/S979C, L339C/V982C, F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment 76 ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Phe343Cys ABCB1 p.Met986Cys Fig. 1D shows that a product with reduced mobility on SDS-PAGE gels was present when mutants F343C/M986C, G346C/G989C, and P350C/ S993C were treated with oxidant. Login to comment 77 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys The highest level of cross-linking occurred in mutant P350C/S993C, since most of the protein migrated with reduced mobility after treatment with oxidant (Fig. 1D, lane 14). Login to comment 78 ABCB1 p.Leu339Cys ABCB1 p.Val982Cys ABCB1 p.Phe336Cys ABCB1 p.Ser979Cys No cross-linked product was observed for mutants F336C/S979C and L339C/V982C. Login to comment 80 ABCB1 p.Leu339Cys ABCB1 p.Phe343Cys ABCB1 p.Val991Cys ABCB1 p.Phe335Cys ABCB1 p.Leu976Cys ABCB1 p.Ser979Cys ABCB1 p.Phe983Cys ABCB1 p.Ser351Cys ABCB1 p.Ala987Cys We also tested mutants F335C/L976C, L339C/S979C, F343C/F983C, G347C/A987C, and S351C/ V991C for cross-linking since they were predicted to lie on opposing faces of TM6 and TM12 modeled in a right-handed coiled-coil. Login to comment 82 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys Effect of Nucleotides and Drug Substrates on Cross-linking-An interesting observation was that the amount of cross-linking seen in mutant L332C/L975C in whole cells (9) varied with the metabolic state of the transfected cells. Login to comment 84 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys A possible explanation was that cross-linking of mutant L332C/L975C was promoted by the presence of ATP. Login to comment 91 ABCB1 p.Phe336Ser ABCB1 p.Phe343Met ABCB1 p.Leu339Val ABCB1 p.Pro350Ser The paired residues Phe-336/Ser-979, Leu-339/Val-982, Phe-343/Met-986, Gly-346/Gly-989, and Pro-350/Ser993 predicted to be close to each other (C, shaded circles) were replaced with cysteine and the mutant P-glycoproteins expressed in HEK 293 cells. Login to comment 96 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys pressing mutant L332C/L975C were cross-linked in the presence of nucleotides. Login to comment 99 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys These results suggest that cross-linking between L332C and L975C occurred during ATP hydrolysis. Login to comment 100 ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Phe343Cys ABCB1 p.Met986Cys The effect of nucleotides on cross-linking was also tested on mutants F343C/M986C, G346C/G989C, and P350C/S993C. Login to comment 103 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Phe343Cys ABCB1 p.Met986Cys To test the effect of drug substrates, cross-linking of mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/ S993C was done in the presence of verapamil, cyclosporin A, vinblastine, or colchicine. Login to comment 104 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys No cross-linked product was observed for mutant L332C/L975C (Fig. 3A). Login to comment 105 ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Phe343Cys ABCB1 p.Met986Cys By contrast, all the drug substrates were effective in blocking cross-linking of mutants F343C/M986C and G346C/G989C (Fig. 3, B and C), but were less effective in preventing cross-linking of mutant P350C/S993C (Fig. 3D). Login to comment 106 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys In mutant P350C/S993C, verapamil, cyclosporin A, and vinblastine were more effective than colchicine in inhibiting cross-linking. Login to comment 107 ABCB1 p.Leu339Cys ABCB1 p.Val982Cys ABCB1 p.Phe336Cys ABCB1 p.Ser979Cys Mutants S979C/F336C or L339C/V982C did not yield any cross-linked product even in the presence of ATP or drug substrates (data not shown). Login to comment 108 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Phe343Cys ABCB1 p.Met986Cys Effect of Cross-linking on Drug-stimulated ATPase Activity- Mutants L332C/L975C, F343C/M986C, G346C/G989C, and P350C/S993C were still active since they retained about 90, 30, 10, and 70%, respectively, of the verapamil-stimulated ATPase activity of the Cys-less P-glycoprotein. Login to comment 109 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Phe343Cys ABCB1 p.Met986Cys Cross-linking of mutants F343C/M986C, G346C/G989C, and P350C/S993C, but not L332C/L975C, was reversed by treatment with dithiothreitol (Fig. 4A). Login to comment 111 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys Mutant P350C/S993C was tested because it exhibited the greatest degree of cross-linking (Fig. 1D). Login to comment 112 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys In addition, mutant P350C/S993C had the highest activity of those mutants whose cross-linked products were sensitive to dithiothreitol. Login to comment 113 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys Membranes prepared from HEK 293 cells expressing Cys-less or mutant P350C/S993C P-glycoprotein- (His)10 were treated with or without 2 mM copper phenanthroline for 10 min at 37 °C. Login to comment 116 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys Fig. 4B (lane 4) shows that the cross-linked mutant P350C/S993C was also efficiently recovered, indicating that the histidine tag remained accessible after cross-linking. Login to comment 119 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys Mutant P350C/S993C, however, showed approximately 75% reduction of the verapamil-stimulated ATPase activity after cross-linking. Login to comment 121 ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys This result suggested that dithiothreitol broke the disulfide bond between residues P350C and S993C and allowed movement to occur between TM6 and TM12 during drug-stimulated ATPase activity. Login to comment 124 ABCB1 p.Leu339Cys ABCB1 p.Val982Cys ABCB1 p.Phe336Cys ABCB1 p.Ser979Cys Cross-linking was not observed between F336C/S979C or L339C/V982C, even in the presence of ATP or drug substrates FIG. 2. Login to comment 126 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Ser993Cys ABCB1 p.Phe343Cys ABCB1 p.Met986Cys Membranes prepared from HEK 293 cells expressing mutants L332C/L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 5 mM ATP, 5 mM ATP plus 0.2 mM sodium vanadate, 5 mM ADP, 5 mM ADP plus 0.2 mM sodium vanadate, or 5 mM AMP-PNP. Login to comment 132 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Ser993Cys ABCB1 p.Phe343Cys ABCB1 p.Met986Cys Membranes prepared from HEK 293 cells expressing mutants L332C/ L975C (A), F343C/M986C (B), G346C/G989C (C), and P350/S993C (D) were treated without (-) or with (ϩ) 2 mM (A) or 0.2 mM (B-D) copper phenanthroline for 10 min at 37 °C in the presence of 1 mM verapamil, 0.1 mM vinblastine, 50 ␮M cyclosporin A, or 5 mM colchicine. Login to comment 140 ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys ATP hydrolysis rather than nucleotide binding was responsible for cross-linking between L332C/ L975C. Login to comment 142 ABCB1 p.Leu975Cys ABCB1 p.Leu975Cys ABCB1 p.Leu332Cys ABCB1 p.Leu332Cys The observation that ATP hydrolysis promoted cross-linking between L332C/L975C suggests that inhibition of cross-linking of L332C/L975C in whole cells by verapamil or vinblastine occurred indirectly through depletion of intracellular ATP. Login to comment 144 ABCB1 p.Gly346Cys ABCB1 p.Gly989Cys ABCB1 p.Pro350Cys ABCB1 p.Ser993Cys ABCB1 p.Phe343Cys ABCB1 p.Met986Cys Drug substrates inhibited cross-linking of mutants F343C/ M986C, G346C/G989C, and P350C/S993C. Login to comment