FUNCTIONAL ANALYSIS OF TRANSPORTER LIGAND INTERACTIONS

生物胺类神经递质多巴胺、去甲肾上腺素和5-羟色胺的质膜转运体具有特殊的治疗意义，因为这些分子泵是精神刺激剂(如可卡因、苯丙胺)和抗抑郁药物(如氟西汀、丙咪嗪)的主要作用部位。尽管目前对转运蛋白如何工作或药物如何与其相互作用知之甚少，但最近对克隆转运蛋白的电生理研究通过证明这些转运蛋白介导了几种离子电流，从而提供了对其功能状态的敏感读数，从而提高了我们的理解。人类多巴胺转运体(HDAT)中识别的电流包括反映底物运动的运输电流、与钠结合相关的瞬时电流和紧张性泄漏电导，该电导可被可卡因类药物和底物阻断。为了在分子水平上了解转运体的作用机制和药物-转运体之间的相互作用，我们开展了小配体对电流的影响以及对HDAT电流和转运活性影响的研究。与特定结构状态相关联的配体相互作用的位置将与PPG的其他组件一起建模。我们确定的电生理学和药理学工具将有助于探索在PPG的这个和其他成分中产生的HDAT结构突变的功能后果。由此产生的模型将通过使用酵母HDAT表达系统进行直接生化和生物物理分析来进一步测试，以产生足够用于此类研究的大量蛋白质。定义转运体-配体相互作用的结构和功能不同状态的长期目标将提供对转运蛋白分子机制的洞察，并将指导未来新型治疗药物的开发。

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.