Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein.
Cooperativity between verapamil and ATP bound to the efflux transporter P-glycoprotein.
复制标题
DOI：
10.1016/j.bcp.2016.08.013
复制
发表时间：
2016-10-15
影响因子：
5.8
通讯作者：
Roberts AG
中科院分区：
文献类型：
--
作者：
Ledwitch KV;Gibbs ME;Barnes RW;Roberts AG
P-糖蛋白转运体(P-gp)通过构象变化和三磷酸腺苷(ATP)的水解将多种化学成分的药物从细胞中排出，在药物处置中起着核心作用。已知许多药物能激活Pgp的ATP水解酶，但ATP与药物结合之间的偶联作用尚不清楚。心血管药物维拉帕米是研究最广泛的PGP底物之一，因此是研究药物诱导PGP ATPase激活的理想药物。如前所述，在饱和的ATP浓度下，维拉帕米诱导的PGP介导的ATP水解动力学是双相的。然而，当ATP浓度低于饱和时，维拉帕米诱导的ATPase激活动力学变得单相。为了进一步了解动力学行为中的这种转换，用一组维拉帕米和ATP浓度来检验PGP偶联的ATPase的活性动力学，并用底物抑制方程和动力学拟合软件COPASI进行拟合。FITS结果提示，三磷酸腺苷和维拉帕米之间的协同作用在低浓度和高浓度之间转换。Pgp的荧光光谱显示，维拉帕米与一种不可水解的ATP类似物之间的协同作用导致了Pgp的全球构象明显变化。脂质体中重组的PGP的核磁共振研究表明，维拉帕米和不可水解性的ATP类似物之间的协同作用调节了彼此的相互作用。这些信息被用来建立药物诱导的PGP介导的ATP水解酶激活的构象门控模型。
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 others interactions. This information was used to produce a conformationally-gated model of drug-induced activation of Pgp-mediated ATP hydrolysis.