Drug-stimulated nucleotide trapping in the human multidrug transporter MDR1. Cooperation of the nucleotide binding domains.
The human multidrug transporter (MDR1 or P-glycoprotein) is an ATP-dependent cellular drug extrusion pump, and its function involves a drug-stimulated, vanadate-inhibited ATPase activity. In the presence of vanadate and MgATP, a nucleotide (ADP) is trapped in MDR1, which alters the drug binding properties of the protein. Here, we demonstrate that the rate of vanadate-dependent nucleotide trapping by MDR1 is significantly stimulated by the transported drug substrates in a concentration-dependent manner closely resembling the drug stimulation of MDR1-ATPase. Non-MDR1 substrates do not modulate, whereas N-ethylmaleimide, a covalent inhibitor of the ATPase activity, eliminates vanadate-dependent nucleotide trapping. A deletion in MDR1 (Delta amino acids 78-97), which alters the substrate stimulation of its ATPase activity, similarly alters the drug dependence of nucleotide trapping. MDR1 variants with mutations of key lysine residues to methionines in the N-terminal or C-terminal nucleotide binding domains (K433M, K1076M, and K433M/K1076M), which bind but do not hydrolyze ATP, do not show nucleotide trapping either with or without the transported drug substrates. These data indicate that vanadate-dependent nucleotide trapping reflects a drug-stimulated partial reaction of ATP hydrolysis by MDR1, which involves the cooperation of the two nucleotide binding domains. The analysis of this drug-dependent partial reaction may significantly help to characterize the substrate recognition and the ATP-dependent transport mechanism of the MDR1 pump protein.
Szabó
K
K
National Institute of Haematology and Immunology, Research Group of the Hungarian Academy of Sciences, H-1113 Budapest, Daróczi u. 24, Hungary.
Welker
E
E
Bakos
Müller
M
M
Roninson
I
I
Váradi
A
A
Sarkadi
B
B
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors; D001665:Binding Sites; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000227:Adenine Nucleotides; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D011994:Recombinant Proteins; C085925:calcein AM; D016572:Cyclosporine; D014700:Verapamil; D000251:Adenosine Triphosphatases; D005033:Ethylmaleimide; D005472:Fluorouracil