PHOTORECEPTOR LIGHT MODULATED CHANNEL--MOLECULAR STUDIES

描述（改编自申请人摘要）： 光化学信息转化为电信号， 视网膜的光感受器外节。 电导率的变化 质膜由cGMP激活的离子通道调节。 这些 通道传导钠和钙以记录瞬时以及 背景光水平的变化。 环核苷酸门控通道（CNGC） 是四聚体，并且已经鉴定了两个亚基，α和β。 每个 亚基具有六个跨膜结构域和一个C-末端环核苷酸 结合结构域，其与cGMP-和cAMP-结合结构域同源， 其他蛋白质。 CNGC没有可用的结构，但有关渠道的见解 构象可以从通道的电生理学研究中推导出来， 在异源表达中与定点诱变偶联的功能 系统 申请人将探索配体识别，调查 特别是通过残基F533、K596和D604的配体配位。 这些 在分子模型中，预测这些残基与嘌呤相互作用 基于结合到E.大肠杆菌cAMP调节型 蛋白质CRP 残基将单独突变和组合突变， 确定它们对配体结合的协同作用。 他们还将检查 镍稳定野生型中通道开放的能力， 突变通道 它们将共同表达α和β亚基， 确定β亚基对配体识别的影响， 通道选通 溶剂中所选残基的可及性 结合结构域和它们在配体结合时的构象变化将被 研究使用半胱氨酸扫描诱变与甲硫基磺酸盐 试剂 将检查CNGC的二价渗透性质，以便 更好地了解通道如何调节钠和 钙离子 数据将根据Eyring率拟合至双中心模型 理论 还将讨论B亚基对二价渗透的影响。 考察 该模型可用于预测钙内流的变化， 细胞内cGMP水平的函数。 牛视网膜CNGC的C-末端胞质区域将表达 作为可溶性蛋白质使用细菌表达系统， 配体直接与片段结合。 结合的有限蛋白水解 结构域片段将用于探测载脂蛋白的整体构象 和蛋白质的配体结合形式。 最后，使用高纯度的 蛋白质片段，它们将使结合结构域结晶， 确定配体-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.