BIOCHEMICAL AND FUNCTIONAL CHARACTERISTICS OF THE CONE CNG CHANNEL

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。光感受器环核苷酸门控（CNG）通道在光传导中起着关键作用。虽然我们对视杆细胞内的CNG通道已经有了很好的描述，但我们对视锥通道的了解还很有限，这主要是由于研究哺乳动物视网膜的视锥系统很困难。视锥细胞在中央视觉中起着至关重要的作用，负责色觉。迄今为止，超过80个锥体CNG通道突变与人类各种形式的色盲和进行性锥体营养不良有关。因此，很明显，阐明锥体营养不良的发病机制需要了解通道的正常结构和功能。最近发现的Nrl-/-小鼠的锥体显性视网膜和锥体衍生细胞系661W细胞是研究锥体特异性蛋白的良好模型。本研究的主要目的是利用锥形模型建立锥形CNG通道的生化和功能特征。第一个具体目标是确定锥形CNG通道的相互作用伙伴。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关联将使用共免疫沉淀和GST下拉试验进行研究。酵母双杂交筛选小鼠视网膜cDNA文库和质谱法将用于进一步鉴定锥体通道相互作用的伙伴。第二个具体目标是表征锥形CNG通道复合物和亚基关联的生化特性。利用蓝色原生电泳和SDS凝胶电泳及化学交联分析Nrl-/-视网膜中的锥形CNG通道复合物。通道复合物的亚基关联将通过共免疫沉淀和GST下拉测定来确定。第三个具体目的是评估Ca2+/calmodulin （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细胞中异种共表达，并利用电生理记录技术研究通道功能。CaM与锥体通道亚基的动态结合将在光处理的Nrl-/-视网膜中使用共免疫沉淀和免疫染色进行评估，通道亚基的原位磷酸化将在Nrl-/-视网膜中进行检测。这些研究的发现将大大增加锥体生物学和发病机制的领域，并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.