PHOTORECEPTOR LIGHT MODULATED CHANNEL--MOLECULAR STUDIES

描述(改编自申请人的摘要)：转换 光化学信息转化为电信号发生在 视网膜的光感受器外节。电导随时间变化 质膜受cGMP激活的离子通道调节。这些 通道同时传导钠和钙以记录瞬变以及 灯光级别的背景变化。环核苷酸门控通道(CNGC) 是四聚体，已鉴定出两个亚基，即α和β。每个人 亚基有六个跨膜结构域和一个C末端的环核苷酸 与cGMP和cAMP结合域同源的结合域 其他蛋白质。 CNGC没有可用的结构，但对渠道有深入的了解 构象可以从通道的电生理研究中得出 在异源表达中结合定点突变的功能 系统。申请者将探索配基识别，调查 特别是由残基F533、K596和D604配位的配体。这些 在分子模型中预测残基与嘌呤相互作用。 基于与大肠杆菌cAMP调控结合的cAMP的坐标 蛋白质，C反应蛋白。残基将单独和组合突变为 确定它们对配体结合的协同作用。他们还将检查 镍在野生型和野生型中稳定通道开放的能力 变异频道。它们会将α和β亚基共同表达为 确定β亚基对配基识别的影响 频道选通。选择的残留物的溶剂可及性 结合结构域及其在配体结合时的构象变化 甲硫磺酸盐半胱氨酸扫描诱变研究 试剂。 将考察CNGCs的二价渗透特性，以便 更好地理解通道如何调节钠和水的流动 钙离子。数据将与基于眼环率的两点模型进行拟合 理论。B亚基对二价渗透的影响也将是 检查过了。该模型可用于预测钙离子内流的变化。 细胞内cGMP水平的函数。 牛视网膜CNGC的C末端胞浆区域将被表达 作为一种可溶蛋白质，使用细菌表达系统来测量 与片段直接结合的配基。结合的有限蛋白分解 结构域片段将用于探测载脂蛋白的整体构象 和配体结合形式的蛋白质。最后，使用高度提纯的 蛋白质片段，它们会使结合域结晶，以便 确定了配体-蛋白质相互作用的X射线结构。

DESCRIPTION (Adapted from applicant's abstract): The conversion of photochemical information into electrical signals takes place in photoreceptor outer segments of the retina. The conductance changes across the plasma membrane are regulated by cGMP-activated ion channels. These channels conduct both sodium and calcium to record transient as well as background changes in light levels. Cyclic nucleotide-gated channels (CNGC) are tetrameric and two subunits, alpha and beta, have been identified. Each subunit has six transmembrane domains and a C-terminal cyclic nucleotide binding domain which is homologous to the cGMP- and cAMP-binding domains of other proteins. There are no structures available for CNGCs, but insights about the channel conformation can be derived from electrophysiological studies of channel function coupled with site-directed mutagenesis in a heterologous expression system. The applicants will explore ligand recognition, investigating specifically the ligand coordination by residues F533, K596 and D604. These residues were predicted to interact with the purine in molecular models based on the coordinates of cAMP bound to the E. coli cAMP-regulatory protein, CRP. The residues will be mutated singly and in combinations to determine their concerted effect on ligand binding. They also will examine the ability of nickel to stabilize the channel opening in the wild type and mutant channels. They will co-express the alpha and beta subunits to determine the influence of the beta subunit on ligand recognition and channel gating. The solvent accessibility of selected residues in the binding domain and their conformational changes upon ligand binding will be investigated using cysteine scanning mutagenesis with methanethiosulfonate reagents. The divalent permeation properties of CNGCs will be examined in order to better understand how the channel regulates the flow of both sodium and calcium ions. Data will be fitted to a two-site model based on Eyring rate theory. The effects of the b subunit on divalent permeation will also be examined. The model can be used to predict the changes in calcium influx as a function of cGMP levels in the cell. The C-terminal cytosolic region of the bovine retina CNGC will be expressed as a soluble protein using a bacterial expression system in order to measure ligand binding to the fragment directly. Limited proteolysis of the binding domain fragment will be used to probe the overall conformation of the apo and ligand-bound forms of the protein. Finally, using highly purified protein fragments, they will crystallize the binding domain in order to determine the X-ray structure of the ligand-protein interaction.