Interacting partners of the neuronal glycine transporter GlyT2: identification, validation and characterization

神经递质转运体在突触传递中起着至关重要的作用，它们的活性对于维持神经递质的动态平衡至关重要。甘氨酸是脑干和脊髓中的主要抑制性神经递质。突触甘氨酸水平受两种高亲和力的质膜转运蛋白GlyT1(甘氨酸转运体1)和GlyT2(甘氨酸转运体1)控制。GlyT2是神经元的异构体，对补充突触前终末的细胞质甘氨酸池是必不可少的。GlyT2基因的突变是导致神经功能障碍亢进的主要原因。此外，GlyT2最近已成为治疗疼痛的潜在靶点。因此，对于基础研究和面向应用的研究，重要的是了解影响GlyT2调控的因素，而蛋白质-蛋白质相互作用可能发挥核心作用。到目前为止，关于这种甘氨酸泵的报道只有几个相互作用的伙伴。我们项目的目的是确定、验证和描述GlyT2的一个新的相互作用伙伴。为了确定一系列可能的相互作用因素，我们将使用高分辨率蛋白质组学，包括免疫共沉淀、下拉分析和质谱学。来源材料将来自野生型和GlyT2基因敲除小鼠。推测的相互作用将通过免疫组织化学共定位实验、邻近连接试验和相互免疫共沉淀来验证。最后，一些有效的相互作用因子将进行生理学表征；它们与GlyT2的相互作用的影响将在运输研究中得到解决，包括分析[~3H]甘氨酸摄取和转运体介导的电流的电生理记录。总而言之，我们的结果将导致对GlyT2分子组织的更好的看法，从而有助于解决GlyT2可能作为治疗靶点的问题。此外，我们的数据将导致关于抑制性突触的多组分性质的假设，这将需要超出本提案范围的进一步研究。

Neurotransmitter transporters play a crucial role in synaptic transmission, and their activity is essential for maintaining neurotransmitter homeostasis. Glycine is the major inhibitory neurotransmitter in the brainstem and spinal cord. Synaptic glycine levels are controlled by two high-affinity electrogenic plasma membrane transporters, namely GlyT1 (glycine transporter 1) and GlyT2. GlyT2 is the neuronal isoform and essential for replenishing the cytoplasmic glycine pool in the presynaptic terminals. Mutations in the GlyT2 gene are a major cause of the neurologic disorder hyperekplexia. Moreover, GlyT2 has recently emerged as a potential target for pain treatment. With regard to both basic and application-oriented research, it is thus important to understand the factors that contribute to GlyT2 regulation, and protein-protein interactions likely play a central role. Only a few interacting partners have thus far been reported for this glycine pump. The aim of our project is to identify, verify, and characterize a novel interacting partners of GlyT2. To identify a comprehensive set of putative interactors, we will employ high-resolution proteomics involving co-immunoprecipitation, pull-down assays, and mass spectrometry. Source material will be obtained from wild-type and GlyT2 knockout mice. Putative interactors will be validated via immunohistochemical colocalization experiments, proximity ligation assays, and reciprocal co-immunoprecipitations. Finally, some validated interactors will undergo physiological characterization; the effect of their interaction with GlyT2 will be addressed in transport studies comprising analysis of [3H]glycine uptake and electrophysiological recordings of transporter-mediated currents. Collectively, our results will lead to a better view of the molecular organization of GlyT2 and, thereby, contribute to the issue in as much GlyT2 may serve as a therapeutic target. Moreover, our data will lead to hypotheses about the multi-component nature of inhibitory synapses that will require further study beyond the scope of this proposal.