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

描述(申请人提供)：多巴胺转运体(DAT)在调节脑多巴胺(DA)的稳态中起着至关重要的作用。通过对DA的再摄取，DAT具有两个重要功能：终止多巴胺能终末的突触传递和补充囊泡型DA池。除了摄取或直接转运外，DAT还可以释放DA。这个过程被称为反向运输或外排，是强效和高度上瘾的精神刺激剂，如苯丙胺及其类似物，用来增加大脑激励和奖赏区域细胞外DA水平的机制。长期以来，人们一直认为DA神经元通过胞吐和非胞吐过程来释放DA。然而，生理信号或精神刺激剂(如苯丙胺)通过DAT诱导DA释放的确切机制仍然是一个复杂且尚未完全了解的研究领域。因此，研究影响DAT反向转运的基本机制(S)对于理解DA调节的基本方面以及与精神刺激剂的治疗使用和滥用相关的DA相关脑疾病的临床干预都是至关重要的。我们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，并阐明精神刺激剂的分子作用。这一应用是基于我们最近的发现，即G蛋白的β-upsilon亚单位(Gbetagamma)与DAT结合并调节转运蛋白的活性。这种效应在培养的细胞、脑突触小体和活体中都得到了证实。更重要的是，Gbetagamma的激活促进了DAT介导的DA外流，而Gbetagamma的抑制则减弱了苯丙胺诱导的DA外流。最后，在体内，Gbetagamma的激活增强，而Gbetagamma的抑制则降低苯丙胺诱发的运动活性。基于这些初步数据，这一建议的中心假设是DAT和β-usilon亚基之间的相互作用通过DAT促进DA的释放，并参与苯丙胺的作用。在这个提案中，我们将：1)确定DAT中的Gbetagamma相互作用位点(S)及其在转运体调节中的作用；2)检验Gbetagamma参与DAT介导的DA外流的假说；3)检验Gbetagamma参与苯丙胺体内作用的假说。这里建议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反向运输的贡献。苯丙胺诱导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，我们提出的研究将不仅定义Gbetagamma亚基在苯丙胺成瘾特性中所起的作用，而且随着我们目前对生理DAT反向运输背后的分子细节的理解，Gbetagamma亚基对DA动态平衡的贡献也将被定义。我们研究计划的长期目标是确定可用于治疗包括药物成瘾在内的神经精神障碍的新的治疗靶点。

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.