Crystallization of neurotransmitter transporter homologs

突触神经传递需要精确控制神经递质的释放和重新摄取。这个 重摄取是由突触前质膜上的特定转运蛋白完成的。这些 转运体从突触间隙重新摄取神经递质，从而使前和 突触后受体。转运蛋白包括依赖Na+/CI的5-羟色胺转运蛋白(SERT)， 多巴胺转运体(DAT)和去甲肾上腺素转运体(NET)，神经递质的所有成员- 钠转运体(NSS)家族。除了它们在正常神经传递中的关键作用外，这些蛋白质 直接与广泛处方的抗抑郁药(如氟西汀(百忧解)、丙咪嗪和 帕罗西汀)或精神刺激剂(如可卡因、安非他明和摇头丸)，所有这些都抑制 神经递质重摄取。尽管它们在生理、药理和临床上具有重要意义，但没有 任何NSS蛋白质都可以获得高分辨率结构信息，部分原因是缺乏系统 哺乳动物膜蛋白的过度表达。为此，我们超量表达并纯化了7个中的5个 选定的哺乳动物神经递质转运体的细菌同系物，毫克量。我们 已确定其中一种是稳定的，单分散在一些洗涤剂中，因此适合于 结晶。该蛋白质与人类转运蛋白有20%-25%的序列同源性。我们建议 生长细菌NSS蛋白的二维晶体并测定其~10A分辨率的结构 使用低温电子显微镜。这张图应该揭示蛋白质的总体结构和它的 膜中的低聚状态。我们还建议将三维蛋白质结晶到~3A 分辨率，这些晶体将被用来解决其高分辨率结构使用X射线结晶学在 项目的下一阶段。

Synaptic neurotransmission requires precise control of the release and reuptake of neurotransmitters. The reuptake is carried out by specific transporter proteins from the presynaptic plasma membrane. These transporters reuptake the neurotransmitters from the synaptic cleft, thereby inactivating both pre- and postsynaptic receptors. The transporters include the Na+/CI-dependent, serotonin transporter (SERT), dopamine transporter (DAT) and norepinephrine transporter (NET), all members of the neurotransmitter- sodium symporter (NSS) family. In addition to their key role in normal neurotransmission, these proteins interact directly with the widely prescribed antidepressants (e.g. fluoxetine (Prozac), imipramine and paroxetine) or psychostimulants (e.g. cocaine, amphetamine and MDMA (ecstasy)), all of them inhibit the neurotransmitter reuptake. In spite of their critical physiological, pharmacological and clinical importance, no high-resolution structural information is available for any NSS protein, partially due to the lacking of systems of overexpressing mammalian membrane proteins. To this end, we overexpressed and purified 5 out of 7 selected bacterial homologues of the mammalian neurotransmitter transporters in milligram quantities. We have identified one of them to be stable and monodisperse in a number of detergents and, thus, suitable for crystallization. The protein shares 20-25% sequence identity with the human transporters. We propose to grow two-dimensional crystals of this bacterial NSS protein and to determine its structure at ~10 A resolution using cryo-electron microscopy. This map should reveal the general architecture of the protein and its oligomeric state in the membrane. We also propose to crystallize the protein in three-dimensions to ~3 A resolution, and the crystals will be used to solve its high resolution structure using X-ray crystallography in the next stage of the project.