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Papers In Press, published online ahead of print August 22, 2001
J. Biol. Chem, 10.1074/jbc.C100467200
Submitted on August 16, 2001
Revised on August 21, 2001
Accepted on August 21, 2001
Department of Medicine, University of Toronto, Toronto, Ontario M5S 1A8
Corresponding Author: david.clarke{at}utoronto.ca
The human multidrug resistance P-glycoprotein (P-gp) interacts with a broad range of compounds with diverse structures and sizes. There is considerable evidence indicating that residues in transmembrane segments (TM) 4, 5, 6 and 10, 11 and 12 form the drug-binding site. We attempted to measure the size of the drug-binding site by using thiol-specific methanethiosulfonate (MTS) cross-linkers containing spacer arms of 2 to 17 atoms. The majority of these cross-linkers were also substrates of P-gp since they stimulated ATPase activity (2.5 to 10.1-fold). Thirty six P-gp mutants with pairs of cysteine residues introduced into TM segments 4-6 and 10-12 were analyzed after reaction with 0.2 mM MTS cross-linker at 4 oC. The cross-linked product migrated with lower mobility than native P-gp in SDS gels. Thirteen P-gp mutants were cross-linked by MTS cross-linkers with spacer arms of 9–25 Å. Vinblastine and cyclosporin A inhibited cross-linking. The emerging picture from these results and other studies is that the drug-binding domain is large enough to accommodate compounds of different sizes and that the drug-binding domain is "funnel" shaped - narrow at the cytoplasmic side, at least 9-25 Å in the middle and wider still at the extracellular surface.
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