Drug-stimulated nucleotide trapping in the human multidrug transporter MDR1: Cooperation of the nucleotide binding domains

Katalin Szabó, E. Welker, E. Bakos, M. Müller, Igor Roninson, A. Váradi, B. Sarkadi

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Abstract

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 (Δ 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.

Original languageEnglish
Pages (from-to)10132-10138
Number of pages7
JournalJournal of Biological Chemistry
Volume273
Issue number17
DOIs
Publication statusPublished - Apr 24 1998

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Nucleotides
Vanadates
Adenosine Triphosphate
Pharmaceutical Preparations
Adenosine Triphosphatases
Substrates
P-Glycoprotein
Pumps
Ethylmaleimide
Methionine
Adenosine Diphosphate
Lysine
Substance-Related Disorders
Carrier Proteins
Hydrolysis
Extrusion
Amino Acids
Mutation
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

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abstract = "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 (Δ 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.",
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T1 - Drug-stimulated nucleotide trapping in the human multidrug transporter MDR1

T2 - Cooperation of the nucleotide binding domains

AU - Szabó, Katalin

AU - Welker, E.

AU - Bakos, E.

AU - Müller, M.

AU - Roninson, Igor

AU - Váradi, A.

AU - Sarkadi, B.

PY - 1998/4/24

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