Coupling of organic anion transport to the glutamate gradient by OATs and OATPs

有机阴离子转运蛋白（OAT）和功能相关的有机阴离子转运肽（OATP）家族转运广泛的生物和非生物重要分子进出细胞。它们运输激素、代谢物、废物和许多药物，包括阿司匹林、抗生素、抗癌、胆固醇和降血压药物。它们介导药物进入体内的肠道吸收，将这些药物转运到靶细胞中，并最终通过肾脏和肝脏将这些药物从体内清除。例如，一些药物不能与葡萄柚汁一起服用的原因是它会抑制特定的OATP。本项目将研究这些转运蛋白如何从化学梯度中获得所需的能量，以承担其基本的生物学和药理学作用。OAT和OATPs可以将物质转运到细胞内外。如果你想将药物泵入细胞，双向运输效率很低（因为进来的东西可以直接再出去）。然而，通过将转运蛋白活性与第二底物偶联，可以在一个方向上介导药物的转移（因为感兴趣的物质被吸收，但不能再次出去，因为第二底物正在使用转运蛋白）。为此，第二底物在细胞内的浓度必须高于所讨论的物质。细胞内的谷氨酸浓度比血浆中高50-100倍，我们发现OAT 4和OATP 2B 1转运谷氨酸。因此，谷氨酸梯度可驱动OAT 4和OATP 2B 1转运的药物和其他分子的摄取。类似于大坝后面储存的水的释放，当谷氨酸从细胞中释放时，储存在梯度中的势能可以被捕获并用于驱动药物的摄取。有多种OAT和OATP转运蛋白，其中许多转运相同的分子。如果这些转运蛋白中的一些与谷氨酸梯度偶联，而另一些没有，那么即使它们转运相同的底物，它们也会以不同的方式起作用，并在体内发挥不同的作用。与谷氨酸梯度偶联的那些（例如OAT 4）将介导药物和激素的摄取，而那些不偶联的则更可能介导外排。该项目的第一个目标是确定OAT和OATP家族的其他成员转运谷氨酸。OAT 4、OATP 2B 1或任何其他OAT/OATP转运谷氨酸的事实并不一定意味着其活性将与谷氨酸梯度偶联;这将取决于谷氨酸转运的动力学、其他底物的存在和谷氨酸的亚细胞定位。该项目的第二个目标是确定特定底物何时被吸收，然后在人类细胞中交换哪些细胞内分子。如果转运蛋白的活性取决于生物膜两侧多种不同底物的浓度，则很难预测转运蛋白的功能。当在上皮屏障的同一膜或顶膜和基膜中存在针对相同底物的不同转运蛋白时，情况甚至更加复杂。该项目的第三个目标是开发一个基于生理学的数学模型，以了解这些转运蛋白如何工作。这将使我们能够预测它们将如何共同发挥作用，以及这将如何影响其内源性底物和治疗剂的转运。OAT和OATP家族发挥着重要的生物学作用，对于我们理解正常细胞功能和药物药理学非常重要。该项目探讨了与其他OAT/OATP底物相比具有较大跨膜梯度的谷氨酸盐驱动这些转运蛋白活性的程度，并提供了数学模型，这将有助于我们解释和预测它们在整个身体中的功能。

The organic anion transporter (OAT) and functionally related organic anion transporting peptide (OATP) families transport a wide range of biologically and pharmacologically important molecules into and out of cells. They transport hormones, metabolites, waste products and many drugs including aspirin, antibiotics, anticancer, cholesterol and blood pressure lowering drugs. They mediate the intestinal absorption of drugs into the body, the transport these drugs into target cells and finally the removal of these drugs from the body via the kidneys and liver. For example, the reason that some medications cannot be taken with grapefruit juice is that it inhibits specific OATPs. This project will investigate how these transporters obtain the energy they require from chemical gradients to undertake their essential biological and pharmacological roles.OAT and OATPs can transport substances both into and out of cells. If you want to pump a drug into a cell, transporting in both directions is inefficient (as what comes in can go straight out again). However, by coupling transporter activity to a second substrate it is possible to mediate transfer of the drug in one direction (as the substance of interest is taken up but cannot get out again because the second substrate is using the transporter). For this to work the second substrate must have a higher concentration within the cell than the substance in question. Glutamate concentrations within the cell are 50-100 x higher than in plasma and we have found that OAT4 and OATP2B1 transport glutamate. Thus the glutamate gradient could drive uptake of drugs and other molecules transported by OAT4 and OATP2B1. In an analogous manner to the release of water stored behind a dam, when the glutamate is released from the cell the potential energy stored in the gradient can be captured and used to drive the uptake of drugs.There are multiple OAT and OATP transporters many of which transport the same molecules. If some of these transporters are coupled to the glutamate gradient, and some are not, then even though they transport the same substrates they will act in different ways and play different roles in the body. Those being coupled to the glutamate gradient (e.g. OAT4) would mediate uptake of drugs and hormones while those which are not would be more likely to mediate efflux. The first aim of this project will be to determine which other members of the OAT and OATP families transport glutamate. The fact that OAT4, OATP2B1 or any other OAT/OATP, transports glutamate does not necessarily mean that its activity will be coupled to the glutamate gradient; this will depend on the kinetics of glutamate transfer, the presence of other substrates and the subcellular localisation of glutamate. The second aim of this project will be to determine when a specific substrate is taken up, then what intracellular molecules is it exchanged for in human cells. It can become difficult to predict how a transporter will function if its activity depends on the concentrations of multiple different substrates on two sides of a biological membrane. It is even more complicated when there are different transporters for the same substrates in the same membrane or in apical and basal membranes of an epithelial barrier. The third aim of this project is to develop a physiologically based mathematical model of how these transporters work. This will allow us to predict how they will function together and how this will affect the transport of their endogenous substrates and of therapeutic agents. The OAT and OATP families play essential biological roles and are important for our understanding of normal cellular function and drug pharmacology. This project explores the extent to which glutamate, which has a large trans-membrane gradient compared to other OAT/OATP substrates, drives the activity of these transporters and provide mathematical models which will help us explain and predict their function throughout the body.