Structural characterization of a human neurotransmitter transporter

描述(申请人提供)：神经元之间信号的调节取决于神经递质释放的频率和突触中存在的神经递质的数量。为随后的神经递质释放事件重置突触的状态需要有效的信号终止。突触前神经元和神经胶质细胞对神经递质分子的再摄取依赖于膜包埋的转运蛋白。这些酶将神经递质从细胞外空间输送到细胞内，因此形成了浓度梯度。为了给这一能量不利的过程提供动力，这些转运体还将钠离子沿浓度梯度向下移动，进入细胞。虽然神经递质转运体直接调节焦虑和寻求奖赏等心理状态在人类中已经得到证实，但临床上相关的拮抗剂对底物识别和抑制的机制知之甚少。由于缺乏任何真核神经递质转运体的原子分辨结构信息，这些努力一直受到阻碍。本申请详细介绍了表达和纯化一种用于结晶和通过X射线衍射确定结构的人类神经递质转运蛋白的策略。这项应用的主要目标是揭示神经递质转运体与神经递质和拮抗剂复合体的三维结构。这一进展将提供结合位点的位置，并产生可测试的假说，以探索转运蛋白如何特异性识别其底物并被拮抗剂抑制。 与公共健康相关：精神活性药物和抗抑郁药被认为作用于位于大脑神经细胞中的神经递质转运体。这些转运体通常会将神经递质吸收回神经细胞，这种活动的不当调节可能会导致药物成瘾和心理障碍。这一应用旨在更好地了解药物是如何通过滥用药物和治疗药物影响神经递质转运体的，希望能开发出更有效的化合物。

DESCRIPTION (provided by applicant): The regulation of signaling between neurons depends on the frequency of neurotransmitter release and quantity of neurotransmitter present in the synapse. Efficient termination of signaling is required for resetting the state of the synapse for a subsequent event of neurotransmitter release. The reuptake of neurotransmitter molecules by presynaptic neurons and glial cells depends on membrane-embedded transporter proteins. These enzymes transport neurotransmitters from the extracellular space into the cell and therefore up a concentration gradient. To power this energetically unfavorable process, these transporters also move sodium ions down their concentration gradient and into the cell. Although it is well-established in humans that neurotransmitter transporters directly regulate psychological states such as anxiety and reward-seeking, much less is known about the mechanisms of substrate recognition and inhibition by clinically relevant antagonists. These efforts have been hampered by the absence of atomic-resolution structural information for any eukaryotic neurotransmitter transporters. This application details strategies to express and purify a human neurotransmitter transporter for crystallization and structural determination via x- ray diffraction. The primary goal of this application is to uncover the three-dimensional structure of a neurotransmitter transporter in complex with neurotransmitter and antagonists. This advance will provide the locations of binding sites and generate testable hypotheses to probe how transporters specifically recognize their substrates and are inhibited by antagonists. PUBLIC HEALTH RELEVANCE: Psychoactive drugs and antidepressants are thought to act on neurotransmitter transporters located in neuronal cells in the brain. These transporters normally absorb neurotransmitters back into neuronal cells, and misregulation of this activity can lead to drug addiction and psychological disorders. This application seeks to better understand how drugs affect neurotransmitter transporters by both abused and therapeutic drugs in the hope that more effective compounds can be developed.