VISUAL ADAPTATION IN THE VERTEBRATE RETINA

描述(申请人提供)：脊椎动物视网膜是一种高度有序的 通过缝隙连接进行电耦合影响的神经元网络 视网膜功能的各个方面。它的每一种不同类型的细胞都有缺口 与相邻细胞的连接，细胞间的连接通信是 在明暗适应过程中由于伴随而受到深刻的影响 细胞外离子、类维甲酸和 神经递质。许多新的视网膜连接蛋白能够形成缝隙连接 通道已被克隆，半连接通道的发现 为研究其药理作用提供了新的工具。 视网膜细胞上的缝隙连接。考虑到不断增加的 与连接蛋白突变相关的人类疾病，极有可能是 许多原因不明的视网膜疾病最终将与 视网膜中表达的连接蛋白的分子结构异常 神经元和神经胶质细胞。显然，更好地了解 视网膜细胞中表达的连接蛋白及其功能属性，以及 它们在脊椎动物视网膜复杂的微电路中扮演着重要的角色。 本申请的主要目的是使用分子， 电生理学和成像技术来识别 视网膜神经元和神经胶质细胞，并表征其电学和 它们的缝隙连接通道和半通道的药理特性 非洲爪哇卵母细胞和转基因细胞中的视网膜神经元。单细胞聚合酶链反应 技术将被用来识别连接蛋白的亚类 视网膜神经元，并将进行实验以检查电信号 原代视网膜细胞与转基因细胞系之间的偶联 已知的视网膜连接蛋白。神经元半通道的化学门控和电压门控 将与表达视网膜的卵母细胞的半通道特性进行比较 缝隙连接蛋白。结构域交换和定点突变将用于 确定连接蛋白序列的区域，这些区域控制着 半条腿。此外，连接蛋白亚基在结构中的组装 将研究缝隙连接通道，以及维甲酸对通道的调制 将对酸、pH、第二信使和其他试剂进行调查。这个 从这些研究中获得的信息，以及连接蛋白的光调节 表达，将使我们更好地理解缝隙连接通道是如何 生理调节，提供对连接蛋白的身份的洞察力 由特定的视网膜细胞类型表达，并有助于阐明 神经功能中的细胞间直接通讯。

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.