Regulation of Dopamine Transporter Function by G Protein Beta-Gamma Subunits

描述（由申请人提供）：多巴胺转运蛋白（DAT）在脑多巴胺（DA）稳态调节中起着至关重要的作用。通过对DA的再摄取，DAT有两个重要的功能：终止多巴胺能末端的突触传递，以及补充囊泡DA池。除了摄取或直接运输外，DAT还可以发挥释放DA的作用。这一过程被称为反向运输或外排，是强效和高度上瘾的精神兴奋剂（如安非他明及其类似物）使用的机制，以增加大脑动机和奖励区域的细胞外DA水平。人们早就认识到DA神经元通过胞外和非胞外过程释放DA。然而，生理信号或精神兴奋剂（如安非他明）通过DAT诱导DA释放的确切机制仍然是一个复杂且未完全理解的研究领域。因此，研究影响通过DAT反向转运的基本机制对于理解DA调节的基本方面和对与治疗性使用和滥用精神兴奋剂相关的DA相关脑疾病的临床干预至关重要。我们的研究计划的长期目标是识别和表征通过DAT控制DA释放的信号机制，并阐明精神兴奋剂的分子作用。这一应用是基于我们最近发现G蛋白的β upsilon亚基（Gbetagamma）结合DAT并调节转运蛋白活性。这种效应在培养细胞、脑突触体和体内均得到证实。更重要的是，在培养细胞中，激活gbetaggamma促进dat介导的DA外排，而抑制gbetaggamma则减弱安非他明引起的DA外排。最后，激活gbetaggamma增强体内运动活性，而抑制gbetaggamma则降低安非他明诱发的运动活性。基于这些初步数据，本提案的中心假设是DAT和β - upsilon亚基之间的相互作用通过DAT促进DA释放，并参与安非他明的作用。在本提案中，我们将i)确定Gbetagamma在DAT中的相互作用位点及其在转运体调节中的作用，ii)验证Gbetagamma参与DAT介导的DA外排的假设，以及iii)验证Gbetagamma参与安非他明体内作用的假设。本文提出的研究的成功完成将提供DAT- gbetagamma相互作用的详细特征，并清楚地了解其对DAT反向传输的贡献。安非他明诱导DAT逆转的事实表明，在生理条件下，DA也可以通过DAT释放。因此，我们提出的研究不仅将确定伽马亚基在安非他明成瘾特性中的作用，还将确定伽马亚基对多巴胺稳态的贡献，因为我们目前对生理数据逆向运输的分子细节有了更深入的了解。我们的研究计划的长期目标是确定新的治疗靶点，可用于治疗神经精神疾病，包括药物成瘾。

DESCRIPTION (provided by applicant): The dopamine transporter (DAT) plays a crucial role in the regulation of brain dopamine (DA) homeostasis. Through re-uptake of DA, DAT serves two important functions: the termination of synaptic transmission at dopaminergic terminals, and the replenishment of vesicular DA pools. In addition to uptake or direct transport, DAT can also function to release DA. This process, which is referred to as reverse transport or efflux, is the mechanism used by potent and highly addictive psychostimulants, such as amphetamine and its analogues, to increase extracellular DA levels in motivational and reward areas of the brain. It has long being recognized that DA neurons release DA through exocytotic and non-exocytotic processes. However, the exact mechanism by which physiological signals or psychostimulants, such as amphetamine, induce DA release through DAT still remains a complex and not completely understood area of research. Thus, examining the basic mechanism(s) that affect reverse transport through DAT is critical for both understanding fundamental aspects of DA regulation and clinical intervention in DA-related brain disorders associated with the therapeutic use and abuse of psychostimulants. The long-term goal of our research program is to identify and characterize signaling mechanisms that control DA release through DAT, and elucidate the molecular actions of psychostimulants. This application is based on our recent discovery that the beta upsilon subunit of G proteins (Gbetagamma) binds DAT and regulates transporter activity. This effect was demonstrated in cultured cells, brain synaptosomes, and in vivo. More importantly, activation of Gbetagamma promotes DAT-mediated DA efflux, whereas inhibition of Gbetagamma attenuates amphetamine-elicited DA efflux in cultured cells. Finally, activation of Gbetagamma enhances whereas inhibition of Gbetagamma reduces amphetamine-evoked locomotor activity in vivo. Based on these preliminary data, the central hypothesis of this proposal is that the interaction between DAT and beta upsilon subunits promotes DA release through DAT and is involved in the actions of amphetamine. In this proposal we will i) identify the Gbetagamma interaction site(s) in DAT and their role in transporter regulation, ii) test the hypothesis that Gbetagamma is involved in DAT-mediated DA efflux, and iii) test the hypothesis that Gbetagamma is involved in amphetamine's actions in vivo. The successful completion of the studies proposed here will provide a detailed characterization of the DAT-Gbetagamma interaction and a clear understanding of its contribution to DAT reverse transport. The fact that amphetamine induces DAT reversal suggests that DA can also be released through DAT under physiological conditions. Therefore, our proposed studies will define not only the role that Gbetagamma subunits play in the addictive properties of amphetamine, but also the contribution of Gbetagamma subunits to DA homeostasis as we grow our current understanding of the molecular details underlying physiological DAT reverse transport. The long-term goal of our research program is to identify novel therapeutic targets that can be used in the treatment of neuropsychiatric disorders, including drug addiction.