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

点击翻译按钮获取中文摘要

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.