Molecular mechanisms: Dysregulation of monoamine transporters by HIV-1 Tat and cocaine

多巴胺能传递的干扰是HIV-1相关的危险因素 神经认知障碍（HAND）。多巴胺能系统在药物奖赏中起因果作用 以及包括认知在内的大脑功能的调节。前额叶皮层是一个重要的大脑 高级认知功能区，其中去甲肾上腺素转运蛋白（NET）是主要的 稳定突触多巴胺能张力的稳态调节机制。HIV-1达特蛋白 和可卡因协同增加突触多巴胺水平，从而产生 神经认知障碍我们的初步研究结果表明，在体外暴露于重组Tat 1 -86， 抑制多巴胺转运蛋白（DAT）和NET对多巴胺和去甲肾上腺素的再摄取， 达特通过直接的蛋白质-蛋白质相互作用与DAT和NET结合。我们已经证明 Tat诱导的DAT功能抑制是通过结合到 DAT，而不是通过与DA摄取位点相互作用。因此，减弱达特与DAT的结合 预计对生理DA转运的影响最小。事实上，我们最近 研究结果表明，一种新型的喹唑啉系列变构调节剂降低了可卡因的效力， 用于抑制DA摄取和减弱Tat诱导的DA再摄取和可卡因抑制 我们假设达特通过独特的变构调节位点，干扰了 DAT和NET调节网络，通常维持集中的DA或NE运输 并加强可卡因对DAT和NET的作用，导致DA/NE相关的神经精神疾病 功能障碍突出表现在手。我们将（目的1）确定达特在 人NET，并探索达特和可卡因对该转运蛋白变构调节;（目的2） 确定DAT/NET介导的多巴胺能传递在诱导型达特中的致病作用 用快速扫描循环伏安法和全细胞膜片钳法评估转基因小鼠 记录;和（目的3）使用新型变构调节剂进行概念验证研究， 使用集成计算方法确定它们在HAND中治疗应用的潜力 建模、药理学和行为学方法。我们的长期目标是探索新的 针对DAT/NET进行治疗干预以改善神经认知功能障碍的方法 同时滥用可卡因者的手。

Perturbation of dopaminergic transmission is implicated as a risk factor of HIV-1 associated neurocognitive disorders (HAND). Dopaminergic system plays a causal role in drug rewarding and modulation of the brain function including cognition. Prefrontal cortex is an important brain region for higher cognitive function, where norepinephrine transporter (NET) is the primary mechanism of homeostatic regulation of stable synaptic dopaminergic tone. HIV-1 Tat protein and cocaine synergistically increase synaptic dopamine levels, thereby producing neurocognitive impairment. Our initial findings show that in vitro exposure to recombinant Tat1-86 inhibits dopamine and norepinephrine reuptake by dopamine transporter (DAT) and NET and Tat binds to DAT and NET through a direct protein-protein interaction. We have demonstrated that Tat-induced inhibition of DAT function is mediated by binding to allosteric binding site(s) on DAT, not by interacting with the DA uptake site. Accordingly, attenuating Tat binding to DAT would be expected to have minimal influence on physiological DA transport. Indeed, our recent findings show that a novel quinazoline series of allosteric modulators decrease cocaine potency for inhibition of DA uptake and attenuate Tat-induced inhibition of DA reuptake and cocaine binding by DAT. We hypothesize that Tat, via the unique allosteric modulatory sites, perturbs the DAT and NET regulatory network that normally sustains concentrative DA or NE transport and potentiates cocaine’s effect on DAT and NET, resulting in DA/NE-linked neuropsychiatric dysfunction prominently featured in HAND. We will (Aim 1) Identify the binding sites for Tat in human NET, and explore allosteric modulation of this transporter by Tat and cocaine; (Aim 2) determine the pathogenic role of DAT/NET-mediated dopaminergic transmission in inducible Tat transgenic mice by assessment of Fast-scan cyclic voltammetry and whole cell patch clamp recording; and (Aim 3) perform proof of concept studies using novel allosteric modulators to establish their potential for therapeutic application in HAND using integrated computational modeling, pharmacological, and behavioral approaches. Our long-term goal is to explore new ways to target DAT/NET for therapeutic interventions to improve neurocognitive dysfunction of HAND in concurrent cocaine abusers.