REGULATION OF NEURONAL SYNAPTIC COMPONENTS

描述（改编自申请人的摘要）： 建议的研究是确定与形成相关的分子机制， 发育中的鸡纤毛上专门的突触和突触周区域 神经节（CG）神经元。CG神经元表达两类烟碱样受体。 乙酰胆碱受体：由a3、a5、b4和偶尔b2组成的nAChR 亚基集中在突触后膜，和Bgt-nAChR 其含有α 7亚基并定位于突触周。的 研究人员最近发现表明，外源嵌合Bgt-nAChR 经工程改造以包含α 3亚基的长胞质环，但不包含α 3亚基的长胞质环。 相应的环从a5或b4亚基，成为本地化的 突触后膜这些结果被解释为表明，内源性 nAChR复合物也可以使用α 3环进行适当的突触定位。 除了乙酰胆碱受体，CG神经元表达抑制性甘氨酸受体（GlyR）。 GlyR和nAChR聚集在单独的但邻近的突触后神经元中。 成熟CG神经元上的膜区域。这些不同的集群共存于 一个花萼型突触前末梢下的神经元表面。计划中的研究 将研究结果逻辑地扩展到4个目标：（1）识别基本的 在a3环中靶向亚结构域;（2）确定该亚结构域是否 将内源性nAChRs定位于突触;（3）定义 突触后膜的微观异质性和空间组织的 分离nAChR和GlyR簇;和（4）鉴定相互作用的蛋白质 与α 3亚结构域，并可能在靶向nAChR的突触的功能。的 研究将涉及分子和形态学分析，包括逆转录病毒 介导的原位基因转移，共聚焦和电子显微镜，和酵母 双杂交测定。

DESCRIPTION (Adapted from the applicant's abstract): The major goal of the proposed studies is to identify molecular mechanisms relevant to the formation of specialized synaptic and perisynaptic regions on developing chick ciliary ganglion (CG) neurons. CG neurons express two classes of nicotinic acetylcholine receptors: nAChRs composed of a3, a5, b4 and occasionally b2 subunits which are concentrated in the postsynaptic membrane, and Bgt-nAChRs which contain a7 subunits, and are localized perisynaptically. The investigator's recent findings show that exogenous, chimeric Bgt-nAChRs engineered to contain the long cytoplasmic loop of the a3 subunit, but not the corresponding loop from the a5 or b4 subunit, become localized in the postsynaptic membrane. These results are interpreted to suggest that endogenous nAChR complexes may also use the a3 loop for appropriate synaptic localization. In addition to AChRs, CG neurons express inhibitory glycine receptors (GlyRs). GlyRs and nAChRs are clustered in separate, but proximate, postsynaptic membrane regions on mature CG neurons. These distinct clusters coexist on the neuron surface under one calyx-type presynaptic terminal. The planned studies will logically extend the findings in 4 aims: (1) identify the essential targeting subdomain in the a3 loop; (2) determine whether this subdomain localizes endogenous nAChRs to the synapse; (3) define the extent of postsynaptic membrane microheterogeneity and the spatial organization of the separate nAChR and GlyR clusters; and (4) identify the protein that interacts with the a3 subdomain and may function in targeting nAChRs to the synapse. The studies will involve molecular and morphological analyses, including retroviral mediated gene transfer in situ, confocal & electron microscopy, and yeast two-hybrid assays.