MODULATION OF TRIGEMINAL INHIBITION

描述（由申请人提供）：伤害感受神经元中的抑制性突触传递主要由甘氨酸受体（GlyR）介导。最近研究表明，细胞内Ca ~（2+）可显著增强GlyR介导的反应。有很好的证据表明，增强是由一个可扩散的细胞质蛋白（CytoP）介导的。本项目的目标是开发一种新的技术来鉴定CytoP并研究其与三叉神经元中GlyRs的相互作用。我们假设，一个可扩散的CytoP结合GlyR在低细胞内浓度（[Ca 2 +]i），保持GlyR在一个低活性状态。当[Ca 2 +]i升高时，蛋白质迅速从GlyRs解离，导致通道活性增强。为了验证这一假设，我们将结合联合收割机分子生物学，膜片钳和成像技术，以确定这些相互作用的分子细节。将通过双杂交方法分离CytoP。CytoP与GlyR的相互作用将通过同时监测（i）使用膜片钳技术的GlyR介导的电流，（2）使用荧光Ca 2+指示剂的细胞内Ca 2+瞬变和（iii）使用频率共振能量转移（FRET）成像技术的GlyR与细胞质蛋白之间的动态相互作用来确定。然后，我们将扩展这项技术，以确定CytoP和研究三叉神经元中的GlyR-CytoP相互作用。对GlyR调控机制的深入了解将为研究神经元信号传导机制提供重要信息，并可能为口面疼痛的治疗提供新的思路。

DESCRIPTION (provided by applicant): Inhibitory synaptic transmission in nociceptive neurons is largely mediated by glycine receptors (GlyRs). It has been shown recently that intracellular Ca2+ causes a large potentiation GlyR-mediated responses. There is good evidence suggesting that the potentiation is mediated by a diffusible cytoplasmic protein (CytoP). The goal of this project is to develop a new technology to identify the CytoP and study its interactions with GlyRs in trigeminal neurons. We hypothesize that a diffusible CytoP binds to GlyRs at low intracellular concentrations ([Ca2+]i), keeping GlyRs in a low activity state. When [Ca2+]i rises, the protein is rapidly dissociated from GlyRs, resulting in an enhancement of channel activity. To test this hypothesis, we will combine molecular biology, patch-clamp and imaging techniques to determine these interactions in molecular details. The CytoP will be isolated by the two-hybrid method. The interaction of the CytoP with GlyRs will be determine by simultaneously monitor (i) GlyR-mediated currents using the patch clamp technique, (2) intracellular Ca2+ transients using fluorescent Ca2+indicators and (iii) dynamic interactions between GlyRs and cytoplasmic proteins using the frequency resonance energy transfer (FRET) imaging technique. We will then extend this technology to identify CytoP and study the GlyR-CytoP interaction in trigeminal neurons. A better understanding of the mechanism of GlyR modulation will provide important information about the neuronal signaling and may lead to new therapy for orofacial pain.