Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein

详细信息    查看全文

• 作者：Kaitlyn V. Ledwitch ; Morgan E. Gibbs ; Robert W. Barnes ; Arthur G. Roberts ; ^{audie@uga.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：AMPPNP ; adenosine 5&prime ; -(β ; γ-imido)triphosphate ; COPASI ; complex pathway simulator ; DDM ; n-dodecyl-β-d-maltoside ; F0 ; H ; initial fluorescence intensity at the high concentration phase ; F0 ; L ; initial fluorescence intensity at the low concentration phase ; KA ; association constant ; KD ; dissociation constant ; KH ; equilibrium constant at high concentration ; KL ; equilibrium constant at low concentration ; KSV ; Stern-Volmer quenching constant ; L ; ligand ; NTA ; nickel-nitrilotriacetic acid ; PAGE ; polyac
• 刊名：Biochemical Pharmacology
• 出版年：2016
• 出版时间：15 October 2016
• 年：2016
• 卷：118
• 期：Complete
• 页码：96-108
• 全文大小：1579 K

文摘

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.