BIOCHEMICAL AND FUNCTIONAL CHARACTERISTICS OF THE CONE CNG CHANNEL

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。光感受器环核苷酸门控(CNG)通道在光信号转导中起着关键作用。虽然视杆细胞中的CNG通道已经得到了很好的描述，但我们对视锥通道的了解还很有限，这主要是因为在哺乳动物视网膜中研究视锥系统的难度。视锥细胞在中央视觉中起着至关重要的作用，并负责颜色视觉。到目前为止，视锥CNG通道的80多个突变与人类各种形式的色觉减退和进行性视锥营养不良有关。因此，阐明视锥细胞营养不良的发病机制需要了解经络的正常结构和功能。NRL-/-小鼠的视锥优势视网膜和视锥细胞系661W细胞是研究视锥特异蛋白的优秀模型。该方案的主要目的是利用锥体模型建立锥体CNG通道的生化和功能特性。第一个具体目标是确定锥形CNG渠道的互动伙伴。锥体通道和已知与杆状通道相互作用的蛋白质之间的潜在联系将使用免疫共沉淀和GST下拉试验进行研究。酵母双杂交筛选小鼠视网膜cDNA文库和质谱学将被用来进一步鉴定锥体通道相互作用的伙伴。第二个具体目的是表征锥体CNG通道复合体的生化特性和亚单位结合。NRL-/-视网膜中的锥形CNG通道复合体将使用蓝色天然和十二烷基硫酸钠凝胶电泳法以及化学交联法进行分析。通道复合体的亚基结合将通过免疫共沉淀和GST下拉试验来确定。第三个特异性目的是评价钙/钙调蛋白(CaM)和磷酸化在调节锥体CNG通道活性中的作用。通道亚基将在661W细胞中异源共表达，并将利用电生理记录技术研究通道功能。在光处理的NRL-/-视网膜中，将使用免疫共沉淀和免疫染色来评估CaM与视锥通道亚单位的动态结合，并将在NRL-/-视网膜中检测通道亚单位的原位磷酸化。拟议中的研究结果将大大增加锥体生物学和发病机制领域的研究成果，这些见解将促进人类锥体疾病治疗策略的发展。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Though CNG channel in rods has been well characterized, our understanding on the cone channel is quite limited, which is primarily due to the difficulty of investigating cone system in mammalian retinas. Cones play an essential role in central vision and are responsible for color vision. To date, over 80 mutations in cone CNG channel have been linked to various forms of achromatopsia and progressive cone dystrophy in humans. Thus, it is overt that elucidating the pathogenesis of cone dystrophy requires understanding of the channel normal structure and function. The recently characterized cone-dominant retinas of Nrl-/- mice and cone-derived cell line, the 661W cells, are excellent models to study cone-specific proteins. The main goal of this proposal is to establish the biochemical and functional characteristics of cone CNG channel using the cone models. The first specific aim is to identify the cone CNG channel interacting partners. The potential associations between the cone channel and those proteins known to interact with the rod channel will be investigated using co-immunoprecipitation and GST pull-down assay. Yeast two-hybrid screening of a mouse retinal cDNA library and mass spectrometry will be used to further identify the cone channel interacting partners. The second specific aim is to characterize the biochemical properties of the cone CNG channel complex and the subunit association. Cone CNG channel complex in the Nrl-/- retinas will be analyzed using blue native and SDS gel electrophoresis and chemical cross-linking. Subunit association of the channel complex will be determined through co-immunoprecipitation and GST pull-down assay. The third specific aim is to evaluate the role of Ca2+/calmodulin (CaM) and phosphorylation in regulating the cone CNG channel activity. The channel subunits will be heterologously co-expressed in 661W cells and the channel function will be studied using electrophysiological recording techniques. The dynamic binding of CaM to the cone channel subunits will be evaluated in the light-treated Nrl-/- retinas using co-immunoprecipitation and immunostaining and in situ phosphorylation of the channel subunits will be examined in the Nrl-/- retinas. Findings from the proposed studies will add substantially to the field of cone biology and pathogenesis and the insights will facilitate the development of therapeutic strategies for human cone diseases.