Biomaterial Database

Defining the drug-binding site in the human multidrug resistance P-glycoprotein using a methanethiosulfonate analog of verapamil, MTS-verapamil. 2001

T W Loo, and D M Clarke

Canadian Institutes for Health Research Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.

Defining the residues involved in the binding of a substrate provides insight into how the human multidrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell. Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of verapamil (MTS-verapamil) and used it with cysteine-scanning mutagenesis to identify the reactive residues within the drug-binding domain of P-gp. MTS-verapamil stimulated the ATPase activity of Cys-less P-gp and had a K(m) value (25 microM) that was similar to that of verapamil. 252 P-gp mutants containing a single cysteine within the predicted transmembrane (TM) segments were expressed in HEK 293 cells and purified by nickel-chelate chromatography and assayed for inhibition by MTS-verapamil. The activities of 15 mutants, Y118C (TM2), V125C (TM2), S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9), N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11), V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by MTS-verapamil. Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and G984C (TM12), were significantly protected from inhibition by MTS-verapamil by pretreatment with verapamil. Less protection was observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11). These results indicate that residues in TMs 4, 6, 10, 11, and 12 must contribute to the binding of verapamil.

UI	MeSH Term	Description	Entries
D008698	Mesylates	Organic salts or esters of methanesulfonic acid.	Mesilate,Methanesulfonates,Mesilates,Mesylate,Methylenesulfonates
D003545	Cysteine	A thiol-containing non-essential amino acid that is oxidized to form CYSTINE.	Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000251	Adenosine Triphosphatases	A group of enzymes which catalyze the hydrolysis of ATP. The hydrolysis reaction is usually coupled with another function such as transporting Ca(2+) across a membrane. These enzymes may be dependent on Ca(2+), Mg(2+), anions, H+, or DNA.	ATPases,Adenosinetriphosphatase,ATPase,ATPase, DNA-Dependent,Adenosine Triphosphatase,DNA-Dependent ATPase,DNA-Dependent Adenosinetriphosphatases,ATPase, DNA Dependent,Adenosinetriphosphatases, DNA-Dependent,DNA Dependent ATPase,DNA Dependent Adenosinetriphosphatases,Triphosphatase, Adenosine
D001665	Binding Sites	The parts of a macromolecule that directly participate in its specific combination with another molecule.	Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D014700	Verapamil	A calcium channel blocker that is a class IV anti-arrhythmia agent.	Iproveratril,Calan,Cordilox,Dexverapamil,Falicard,Finoptin,Isoptin,Isoptine,Izoptin,Lekoptin,Verapamil Hydrochloride,Hydrochloride, Verapamil
D016296	Mutagenesis	Process of generating a genetic MUTATION. It may occur spontaneously or be induced by MUTAGENS.	Mutageneses
D018432	Drug Resistance, Multiple	Simultaneous resistance to several structurally and functionally distinct drugs.	Drug Resistance, Extensively,Extensively Drug Resistance,Extensively-Drug Resistance,Multidrug Resistance,Multi-Drug Resistance,Extensively Drug Resistances,Extensively-Drug Resistances,Multiple Drug Resistance,Resistance, Extensively Drug,Resistance, Extensively-Drug,Resistance, Multiple Drug
D020168	ATP Binding Cassette Transporter, Subfamily B, Member 1	A 170-kDa transmembrane glycoprotein from the superfamily of ATP-BINDING CASSETTE TRANSPORTERS. It serves as an ATP-dependent efflux pump for a variety of chemicals, including many ANTINEOPLASTIC AGENTS. Overexpression of this glycoprotein is associated with multidrug resistance (see DRUG RESISTANCE, MULTIPLE).	ATP-Dependent Translocase ABCB1,MDR1 Protein,MDR1B Protein,Multidrug Resistance Protein 1,P-Glycoprotein,P-Glycoprotein 1,ABCB1 Protein,ATP Binding Cassette Transporter, Sub-Family B, Member 1,ATP-Binding Cassette, Sub-Family B, Member 1,CD243 Antigen,PGY-1 Protein,1, P-Glycoprotein,ABCB1, ATP-Dependent Translocase,ATP Dependent Translocase ABCB1,Antigen, CD243,P Glycoprotein,P Glycoprotein 1,PGY 1 Protein,Protein, MDR1B,Translocase ABCB1, ATP-Dependent