Neuronal Nicotinic Receptors- Expression and Regulation

描述（由申请人提供）：尼古丁信号传导广泛存在于神经系统中，并影响许多行为和神经病理学。信号传导依赖于烟碱受体，其通常促进钙内流并调节钙依赖性事件。因此，信号传导的结果可能主要取决于受体的位置和相关细胞内成分的身份。最近已经显示PSD-95/SAP 90家族的成员形成与神经元上的烟碱受体相关的突触后PDZ支架。支架不仅有助于介导下游信号传导，而且在支持神经元的突触输入方面发挥重要但未知的作用。初步结果表明，突触后神经配素，EphB 2受体，和快速的活性驱动的SNARE依赖性受体贩运也收敛于突触后神经元，以调节烟碱信号。我们提出了四个具体的目标来追求这些发现：（1）确定突触后PDZ支架如何影响突触输入，并确定可能的中间组件发挥作用。(2)检验假设：突触后神经配素支持烟碱输入独立于PDZ支架，并决定它如何与烟碱受体相互作用。(3)研究促进神经元上烟碱受体快速运输的机制，并评估其对突触信号传导的生理意义。(4)测试EphB 2受体增强烟碱效应的假设，并检查控制神经元中烟碱信号传导的会聚通路的相互依赖性。荧光成像将用于可视化突触组件;转染将用于操纵相互作用的伴侣;电生理分析将用于研究功能后果;生化和分子生物学技术将用于探测相互作用。实验将主要集中在鸡睫状神经节神经元，但也将采用转染的细胞系来定义分子相互作用，和大鼠海马神经元来评估研究结果的一般性。这些结果将为烟碱信号的调控机制提供新的见解，这一点很重要，因为这种信号是普遍存在的，并具有深远的生物医学后果。

DESCRIPTION (provided by applicant): Nicotinic signaling is widespread in the nervous system and influences numerous behaviors and neuropathologies. The signaling depends on nicotinic receptors which often promote calcium influx and regulate calcium-dependent events. The consequence of signaling, therefore, can depend critically both on receptor location and on the identity of associated intracellular components. Recently it has been shown that members of the PSD-95/SAP90 family form a postsynaptic PDZ-scaffold associated with nicotinic receptors on neurons. The scaffold not only helps mediates downstream signaling but also plays an important but unknown role in supporting synaptic input to the neuron. Preliminary results suggest that postsynaptic neuroligin, EphB2 receptors, and rapid activity-driven SNARE-dependent receptor trafficking also converge in the postsynaptic neuron to regulate nicotinic signaling. Four specific aims are proposed to pursue these findings: (1) Determine how postsynaptic PDZ-scaffolds influence synaptic input and identify possible intermediate components exerting the effect. (2) Test the hypothesis: that postsynaptic neuroligin supports nicotinic input independently from PDZ-scaffolds and determine how it interacts with nicotinic receptors. (3) Examine the mechanisms promoting rapid trafficking of nicotinic receptors on neurons and assess their physiological significance for synaptic signaling. (4) Test the hypothesis that EphB2 receptors enhance nicotinic effects and examine the interdependence of convergent pathways controlling nicotinic signaling in neurons. Fluorescence imaging will be used to visualize synaptic components; transfections will be used to manipulate interacting partners; electrophysiological analysis will be used to study functional consequences; and biochemical and molecular biological techniques will be used to probe interactions. Experiments will focus mainly on chick ciliary ganglion neurons but will also employ transfected cell lines to define molecular interactions, and rat hippocampal neurons to assess the generality of the findings. These results will provide new insight into regulatory mechanisms that shape nicotinic signaling, important because the signaling is pervasive and has profound biomedical consequences.