BIOCHEMICAL AND FUNCTIONAL CHARACTERISTICS OF THE CONE CNG CHANNEL

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 光感受器环核苷酸门控（CNG）通道在光转导中起着关键作用。尽管CNG通道在视杆细胞中已经被很好地表征，但我们对锥细胞通道的理解是相当有限的，这主要是由于在哺乳动物视网膜中研究锥细胞系统的困难。视锥细胞在中央视觉中起着至关重要的作用，并负责色觉。迄今为止，锥CNG通道中的超过80个突变已经与人类中的各种形式的色盲和进行性锥营养不良相关联。因此，明确阐明视锥细胞营养不良的发病机制需要了解通道的正常结构和功能。最近表征的Nrl-/-小鼠的视锥优势视网膜和视锥衍生的细胞系661 W细胞是研究视锥特异性蛋白的极好模型。本提案的主要目标是使用锥体模型建立锥体CNG通道的生物化学和功能特性。第一个具体的目的是确定锥CNG通道相互作用的合作伙伴。将使用免疫共沉淀和GST下拉试验研究锥通道和已知与杆通道相互作用的那些蛋白质之间的潜在关联。酵母双杂交筛选小鼠视网膜cDNA文库和质谱将用于进一步确定锥通道相互作用的合作伙伴。第二个具体目标是表征锥CNG通道复合物和亚基缔合的生化性质。将使用蓝色天然和SDS凝胶电泳和化学交联分析Nrl-/-视网膜中的锥状CNG通道复合物。将通过免疫共沉淀和GST下拉试验测定通道复合物的亚基缔合。第三个具体的目的是评估钙/钙调蛋白（CaM）和磷酸化在调节锥CNG通道活性的作用。通道亚基将在661 W细胞中异源共表达，并将使用电生理记录技术研究通道功能。将使用免疫共沉淀和免疫染色在光处理的Nrl-/-视网膜中评价CaM与视锥通道亚基的动态结合，并将在Nrl-/-视网膜中检查通道亚基的原位磷酸化。 从拟议的研究结果将大大增加锥生物学和发病机制的领域和见解将促进人类锥疾病的治疗策略的发展。

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.