P-glycoprotein (P-gp) expressed in a confluent monolayer of hMDR1-MDCKII cells has more than one efflux pathway with cooperative binding sites
P-glycoprotein (P-gp) expressed in a confluent monolayer of hMDR1-MDCKII cells has more than one efflux pathway with cooperative binding sites
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DOI：
10.1021/bi060593b
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发表时间：
2006-12-26
期刊：
影响因子：
2.9
通讯作者：
Bentz, Joe
中科院分区：
文献类型：
--
作者：
Acharya, Poulomi;Tran, Thuy T.;Bentz, Joe
多药耐药转运蛋白P-糖蛋白（P-gp）可外排多种底物，并可受到多种抑制剂或调节剂的影响。许多研究已经在平衡结合或P-gp的ATP酶活性的Michaelis-Menten酶分析的背景下对这些结合相互作用进行了分类。我们的方法是研究P-gp的运输和其抑制使用的hMDR 1-MDCKII细胞的生理相关的融合单层。我们测量的基本速率常数P-gp流出的基板和研究抑制使用成对组合与不同的未标记的基板作为抑制剂。我们目前的P-gp动力学模型只有一个单一的结合位点，因为以前的研究证明，一个单一的底物外排的质量作用动力学是不敏感的是否有一个或多个底物结合和外排位点。在这项研究中，使用这个一个网站的模型，我们发现，与“高”浓度的任何一个基板或抑制剂，基本速率常数拟合独立为每个基板单独定量预测的外排曲线，简单地应用的假设，在“一个网站”的结合是竞争性的。另一方面，在“低”浓度的底物和抑制剂，我们发现没有抑制底物外排，尽管事实上，底物和抑制剂都被很好地外排。这不是过量“空”P-gp分子的影响，因为竞争性外排模型考虑了位点占用。相反，这是定量证据表明底物和抑制剂通过P-gp内的多种途径外排。值得注意的是，将底物浓度增加到“低”浓度以上，导致抑制变得竞争性;即，抑制剂变得有效。这些数据及其分析表明，这些底物的结合必须是合作的，无论是积极的还是消极的。
The multidrug resistance transporter P-glycoprotein (P-gp) effluxes a wide range of substrates and can be affected by a wide range of inhibitors or modulators. Many studies have presented classifications for these binding interactions, within either the context of equilibrium binding or the Michaelis-Menten enzyme analysis of the ATPase activity of P-gp. Our approach is to study P-gp transport and its inhibition using a physiologically relevant confluent monolayer of hMDR1-MDCKII cells. We measure the elementary rate constants for P-gp efflux of substrates and study inhibition using pairwise combinations with a different unlabeled substrate acting as the inhibitor. Our current kinetic model for P-gp has only a single binding site, because a previous study proved that the mass-action kinetics of efflux of a single substrate were not sensitive to whether there are one or more substrate-binding and efflux sites. In this study, using this one-site model, we found that, with "high" concentrations of either a substrate or an inhibitor, the elementary rate constants fitted independently for each of the substrates alone quantitatively predicted the efflux curves, simply applying the assumption that binding at the "one site" was competitive. On the other hand, at "low" concentrations of both the substrate and inhibitor, we found no inhibition of the substrate efflux, despite the fact that both the substrate and inhibitor were being well-effluxed. This was not an effect of excess "empty" P-gp molecules, because the competitive efflux model takes site occupancy into account. Rather, it is quantitative evidence that the substrate and inhibitor are being effluxed by multiple pathways within P-gp. Remarkably, increasing the substrate concentration above the "low" concentration, caused the inhibition to become competitive; i.e., the inhibitor became effective. These data and their analysis show that the binding of these substrates must be cooperative, either positive or negative.