Tran Thuy Thanh, Mittal Aditya, Aldinger Tanya, Polli Joseph W, Ayrton Andrew, Ellens Harma, Bentz Joe
Department of Bioscience & Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104, USA.
Biophys J. 2005 Jan;88(1):715-38. doi: 10.1529/biophysj.104.045633. Epub 2004 Oct 22.
The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized.
人类多药耐药膜转运蛋白P-糖蛋白（P-gp）因其对人类健康和疾病的重要性而受到广泛研究。到目前为止，P-gp转运的动力学分析仅限于稳态米氏方法或房室模型，而这两种方法都无法证明分子机制。确定转运的基本动力学速率常数对于理解P-gp的工作方式至关重要。我们使用的实验系统是过表达P-gp的MDCKII-hMDR1细胞汇合单层。这是一个生理相关的模型系统，并且在不对P-gp进行生化操作的情况下测量转运。米氏质量作用反应用于模拟P-gp转运。在不施加稳态假设的情况下，该反应取决于几个必须同时拟合的参数。使用分层算法，在合理的计算时间内完成了对转运数据的详尽拟合，以找到最适合数据的所有可能参数向量。对于三种P-gp底物（安普那韦、洛哌丁胺和奎尼丁），我们成功拟合了基本速率常数，即药物从顶端膜内层与P-gp结合、药物解离回到顶端膜内层以及药物从P-gp流出到顶端腔室，以及流出活性P-gp的密度。所有三种药物的流出活性P-gp范围重叠，这是拟合过程有效性的一个基准。一个新发现是，对于所有三种药物，与P-gp的结合似乎仅受药物在质膜内层内横向扩散的速率限制。如果P-gp结构如最近的结构研究所示对顶端膜内层的脂质开放，那么这是可以预期的。拟合的动力学参数显示了P-gp对多种外源性物质的流出是如何最大化的。
