MODULATION OF TRIGEMINAL INHIBITION

描述(申请人提供)：伤害性神经元中的抑制性突触传递很大程度上是由甘氨酸受体(GlyRs)介导的。最近的研究表明，细胞内的钙离子对GlyR介导的反应有很大的增强作用。有很好的证据表明，这种增强是由一种可扩散的细胞质蛋白(CytoP)介导的。这个项目的目标是开发一种新的技术来识别细胞色素P，并研究它与三叉神经细胞中GlyRs的相互作用。我们假设，可扩散的CytoP在低细胞内浓度([Ca~(2+)]i)与GlyRs结合，使GlyRs保持低活性状态。当[Ca~(2+)]i升高时，该蛋白迅速从GlyRs上解离，导致通道活性增强。为了验证这一假设，我们将结合分子生物学、膜片钳和成像技术来确定分子细节上的这些相互作用。用双杂交法分离细胞色素P。CytoP与GlyRs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)使用荧光钙指示剂的细胞内钙瞬变，以及(Iii)使用频率共振能量转移(FRET)成像技术的GlyRs与细胞质蛋白质之间的动态相互作用来确定。然后，我们将把这项技术扩展到鉴定CytoP，并研究三叉神经细胞中GlyR-CytoP的相互作用。更好地了解GlyR的调节机制将提供有关神经元信号转导的重要信息，并可能为口面部疼痛的治疗提供新的治疗方法。

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.