title: Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein.
abstract: The P-glycoprotein (Pgp) transporter plays a central role in drug disposition by effluxing a chemically diverse range of drugs from cells through conformational changes and ATP hydrolysis. A number of drugs are known to activate ATP hydrolysis of Pgp, but coupling between ATP and drug binding is not well understood. The cardiovascular drug verapamil is one of the most widely studied Pgp substrates and therefore, represents an ideal drug to investigate the drug-induced ATPase activation of Pgp. As previously noted, verapamil-induced Pgp-mediated ATP hydrolysis kinetics was biphasic at saturating ATP concentrations. However, at subsaturating ATP concentrations, verapamil-induced ATPase activation kinetics became monophasic. To further understand this switch in kinetic behavior, the Pgp-coupled ATPase activity kinetics was checked with a panel of verapamil and ATP concentrations and fit with the substrate inhibition equation and the kinetic fitting software COPASI. The fits suggested that cooperativity between ATP and verapamil switched between low and high verapamil concentration. Fluorescence spectroscopy of Pgp revealed that cooperativity between verapamil and a non-hydrolyzable ATP analog leads to distinct global conformational changes of Pgp. NMR of Pgp reconstituted in liposomes showed that cooperativity between verapamil and the non-hydrolyzable ATP analog modulate each other's interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis.
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602, United States.
authors.forename: Kaitlyn V
authors.initials: KV
authors.lastname: Ledwitch
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602, United States.
authors.forename: Morgan E
authors.initials: ME
authors.lastname: Gibbs
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602, United States.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Barnes
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602, United States. Electronic address: audie@uga.edu.
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Roberts
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000819:agonists* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D002121:Calcium Channel Blockers / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D003198:Computer Simulation; D006868:Hydrolysis / Q000187:drug effects; D008024:Ligands; D008081:Liposomes; D051379:Mice; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014700:Verapamil / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D000889:Anti-Arrhythmia Agents; D002121:Calcium Channel Blockers; D008024:Ligands; D008081:Liposomes; D011994:Recombinant Proteins; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D014700:Verapamil; C507084:Abcb1b protein, mouse
keywords: ABC transporter; Cardiovascular; Cooperativity; Drug transport; Nuclear magnetic resonance (NMR); Nucleotide; adenosine 5′-(β,γ-imido)triphosphate (AMPPNP, PubChem CID: 33113); verapamil hydrochloride (PubChem CID: 62969)