Department of Computational and Systems Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
Department of Cell Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.
Neuropharmacology. 2019 Dec 15;161:107676. doi: 10.1016/j.neuropharm.2019.107676. Epub 2019 Jun 20.
Recent work demonstrated the propensity of dopamine transporters (DATs) to form trimers or higher oligomers, enhanced upon binding a furopyrimidine, AIM-100. AIM-100 binding promotes DAT endocytosis and thereby moderates dopaminergic transmission. Despite the neurobiological significance of these events, the molecular mechanisms that underlie the stabilization of DAT trimer and the key interactions that modulate the trimerization of DAT, and not serotonin transporter SERT, remain unclear. In the present study, we determined three structural models, termed trimer-W238, -C306 and -Y303, for possible trimerization of DATs . To this aim, we used structural data resolved for DAT and its structural homologs that share the LeuT fold, advanced computational modeling and simulations, site-directed mutagenesis experiments and live-cell imaging assays. The models are in accord with the versatility of LeuT fold to stabilize dimeric or higher order constructs. Selected residues show a high propensity to occupy interfacial regions. Among them, D231-W238 in the extracellular loop EL2, including the intersubunit salt-bridge forming pair D231/D232-R237 (not present in SERT) (in trimer-W238), the loop EL3 (trimers-C306 and -Y303), and W497 on the intracellularly exposed IL5 loop (trimer-C306) and its spatial neighbors (e.g. K525) near the C-terminus are computationally predicted and experimentally confirmed to play important roles in enabling the correct folding and/or oligomerization of DATs in the presence of AIM-100. The study suggests the possibility of controlling the effective transport of dopamine by altering the oligomerization state of DAT upon small molecule binding, as a possible intervention strategy to modulate dopaminergic signaling. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
最近的研究表明，多巴胺转运体（DAT）在结合呋喃嘧啶 AIM-100 后倾向于形成三聚体或更高的寡聚体，从而增强其稳定性。AIM-100 的结合促进了 DAT 的内吞作用，从而调节了多巴胺能传递。尽管这些事件具有神经生物学意义，但仍不清楚稳定 DAT 三聚体的分子机制以及调节 DAT 三聚化而不是 5-羟色胺转运体 SERT 三聚化的关键相互作用。在本研究中，我们确定了三个结构模型，分别称为 DAT 三聚体-W238、-C306 和 -Y303，用于 DAT 可能的三聚化。为此，我们使用 DAT 及其结构同源物的结构数据，这些同源物共享 LeuT 折叠，先进的计算建模和模拟，定点突变实验和活细胞成像测定。这些模型与 LeuT 折叠的多功能性一致，能够稳定二聚体或更高阶结构。选定的残基显示出占据界面区域的高倾向。其中，包括二聚体盐桥形成对 D231/D232-R237（不在 SERT 中）的细胞外环 EL2 中的 D231-W238（在三聚体-W238 中）、EL3 环（三聚体-C306 和 -Y303）和细胞内暴露的 IL5 环上的 W497（三聚体-C306）及其空间相邻物（例如 K525）在靠近 C 末端处，在 AIM-100 存在的情况下，计算预测和实验证实它们在 DAT 的正确折叠和/或寡聚化中发挥重要作用。该研究表明，通过改变小分子结合后 DAT 的寡聚化状态，有可能控制多巴胺的有效转运，这可能是一种调节多巴胺能信号的干预策略。本文是题为“神经递质转运体特刊”的一部分。
