FUNCTIONAL ANALYSIS OF TRANSPORTER LIGAND INTERACTIONS

生物胺神经递质多巴胺、去甲肾上腺素和5-羟色胺的质膜转运蛋白具有特别的治疗意义，因为这些分子泵是精神兴奋剂的主要作用部位（例如，可卡因、安非他明）和抗抑郁药物（例如，氟西汀、丙咪嗪安非他酮）。虽然它是知之甚少的转运蛋白如何工作或药物如何与他们相互作用，最近的克隆转运蛋白的电生理学研究表明，这些转运蛋白介导的几个离子电流，提供灵敏的读出他们的功能状态，推进了我们的理解。在人类多巴胺转运蛋白（hDAT）中识别的电流包括反映底物运动的转运电流、与钠结合相关的瞬态电流以及可被可卡因样药物和底物阻断的强直性漏电导。我们已经开始研究小配体如何影响电流和hDAT的转运活性，以了解分子水平上的转运机制和药物-转运蛋白相互作用。与特定结构状态相关的配体相互作用位点将与PPG的其他组分一起建模。我们确定的电生理和药理学工具将是有用的，用于探测在PPG的这个和其他组件中产生的hDAT的结构突变的功能后果。将使用酵母hDAT表达系统通过直接生物化学和生物物理分析进一步测试所得模型，以产生足以用于此类研究的蛋白质量。结构定义功能不同状态的转运蛋白-配体相互作用的长期目标将提供对转运的分子机制的深入了解，并将指导未来新型治疗剂的开发。

The plasma membrane transporters for the biogenic amine neurotransmitters dopamine, norepinephrine and serotonin are of particular therapeutic interest because these molecular pumps are the primary sites of action of psychostimulant (e.g., cocaine, amphetamine) and antidepressant drugs (e.g., fluoxetine, imipramine bupropion). Although it is poorly understood how the transporters work or how drugs interact with them, recent electrophysiological studies of cloned transporters have advanced our understanding by demonstrating that these transporter mediate several ionic currents that provide sensitive readouts of their functional states. Currents identified in the human dopamine transporter (hDAT) include a transport current reflecting substrate movement, transient currents associated with sodium binding, and a tonic leak conductance which can be blocked by both cocaine-like drugs and by substrates. We have embarked on a study of how small ligands affect the currents affect the currents and transport activity of the hDAT in order to learn about transporter mechanisms and drug- transporter interactions at the molecular level. The sites of ligand interaction associated with specific structural states will be modeled in conjunction with other components of the PPG. The electrophysiological and pharmacological tools we identified will be useful for probing the functional consequences of structural mutations of hDAT generated in this and other components of the PPG. The resulting models will be further tested by direct biochemical and biophysical analysis using a yeast hDAT expression system to generate quantities of protein sufficient for such studies. The long term goals of structural defining functionally distinct states of transporter-ligand interaction will provide insight into the molecular mechanisms of transport and will guide future development of novel therapeutic agents.