VISUAL ADAPTATION IN THE VERTEBRATE RETINA

描述（由申请人提供）：脊椎动物视网膜是高度有序的 神经元网络，其中通过缝隙连接的电耦合影响 视网膜功能的各个方面它的各种细胞类型的每一个使差距 与相邻细胞的连接，以及细胞间的连接通讯， 在光适应和暗适应过程中， 细胞外离子、类维生素A和 神经传递素许多能够形成缝隙连接的新视网膜连接蛋白 通道已经被克隆，半连接通道的发现 提供了一个新的工具，研究药理学性质的 视网膜细胞上的差距连接。考虑到越来越多的 与连接蛋白突变相关的人类疾病， 许多不明原因的视网膜疾病最终将与 视网膜中表达的连接蛋白分子结构的畸变 神经元和神经胶质。显然，更好地了解 在视网膜细胞中表达的连接蛋白，它们的功能属性，以及 它们在脊椎动物视网膜的复杂微电路中的重要作用。 本申请的主要目的是使用分子， 电生理和成像技术来识别所使用的连接蛋白 视网膜神经元和神经胶质，并表征电和 它们的缝隙连接通道和半通道的药理学性质， 视网膜神经元，在非洲爪蟾卵母细胞，并在转染的细胞。单细胞PCR 技术将被用来确定亚类的连接蛋白含量， 视网膜神经元，实验将进行检查电 天然视网膜细胞和表达 已知的视网膜连接蛋白神经元半通道的化学和电压门控 将与表达视网膜色素的卵母细胞的半通道特性进行比较， 连接蛋白结构域交换和定点诱变将用于 确定连接蛋白序列的区域， 半通道此外，连接蛋白亚基的组装在形成 缝隙连接通道的研究，以及视黄酸对通道的调节 酸，pH值，第二信使和其他代理人将被调查。的 从这些研究中获得的信息，以及连接蛋白的光调节 表达，将提供一个更好的理解如何间隙连接通道是 调节生理，提供洞察身份的连接蛋白 由特定的视网膜细胞类型表达，并有助于阐明 神经功能中的直接细胞间通讯。

DESCRIPTION (provided by applicant): The vertebrate retina is a highly ordered neuronal network in which electrical coupling through gap junctions influences every aspect of retinal function. Each of its various cell types make gap junctions with neighboring cells, and intercellular junctional communication is profoundly affected during light- and dark-adaptation by virtue of concomitant changes in the extracellular concentrations of ions, retinoids, and neurotransmitters. Many new retinal connexins capable of forming gap-junction channels have been cloned, and the discovery of hemi-junctional channels provides a new tool with which to investigate the pharmacological properties of the gap junction on retinal cells. Considering the ever-increasing number of human diseases associated with connexin mutations, it is highly likely that many retinal disorders of unknown origin will ultimately be linked to aberrations in the molecular structure of the connexins expressed in retinal neurons and glia. Clearly, it is important to gain a better understanding of the connexins expressed in retinal cells, their functional attributes, and their essential role in the complex microcircuitry of the vertebrate retina. The principal goals of the present application are to use molecular, electrophysiological, and imaging techniques to identify the connexins used by retinal neurons and glia, and to characterize the electrical and pharmacological properties of their gap-junctional channels and hemichannels in retinal neurons, in Xenopus oocytes, and in transfected cells. Single-cell PCR techniques will be used to identify the connexin content of subclasses of retinal neurons, and experiments will be performed to examine electrical coupling between native retinal cells and transfected cell lines expressing known retinal connexins. Chemical- and voltage-gating of neuronal hemichannels will be compared with the hemichannel properties of oocytes expressing retinal connexins. Domain swapping and site-directed mutagenesis will be used to determine regions of the connexin sequence that govern the formation of hemichannels. In addition, the assembly of connexin subunits in the formation of gap-junctional channels will be studied, and channel modulation by retinoic acid, pH, second messengers, and other agents will be investigated. The information gained from these studies, and on the photic regulation of connexin expression, will afford a better understanding of how gap-junction channels are regulated physiologically, provide insights into the identity of the connexins expressed by specific retinal cell types, and help to elucidate the role of direct cell-cell communication in neural function.