CONFORMATIONAL ASPECTS OF VISUAL EXCITATION

这项研究的总体目标是阐明其分子基础。 视网膜视杆细胞的视觉兴奋和适应。重点将是 关于钙信号是如何在视力恢复和恢复中被读取和处理的 适应。以下是酶、光谱和 将对杆状外节(ROS)蛋白进行电生理研究 开展了以下工作：(1)恢复素作为一种蛋白的分子作用机制。 视觉中的钙传感器将通过功能重构来定义 钙敏感的鸟苷环化酶活性。除了蛋白质外，还有 恢复素和鸟苷环化酶，是提供钙的关键- 敏感的激活将被提纯和表征。特别是， 承载GDP的转导蛋白α亚基的潜在参与 (t-α-GDP)将被评估。(2)复苏素是否也控制了 蛙的cGMP磷酸二酯酶活性和S-调制素的活性 罗斯？如果是这样的话，恢复的作用地点-它是否直接作用于 光激发视紫红质、视紫红质激酶、转导蛋白或其他成分 -将被划定。(3)视网膜恢复素是在其氨基末端酰化的。 末端由肉豆蔻酸(14：0)或相关的酰基(14：1，14：2， 12：0)。含有这些酰基的重组恢复素将是 准备好了。附着的酰基对钙诱导的影响 将测量恢复素在膜中的插入。的几何学 膜插入的恢复素将被荧光能量描绘出来 转移光谱学。(4)与钙离子中的恢复素结合的蛋白质- 具体方式将通过亲和层析鉴定 回收蛋白-琼脂糖凝胶、共价交联和荧光发射 各向异性光谱。(5)Recovery in的定点突变体将是 为确定不同地区的功能意义而制作的。的 特别感兴趣的是EF手2和3的钙结合部位， EF手1和2之间的疏水缝隙，EF手之间的连接物 3和4，以及高电荷的C-末端尾巴。(6)电生理 天然和突变恢复素以及T-α-GDP影响的研究 关于截短杆的膜电流将进行补充 生化和光谱研究。关于诉讼模式的研究 恢复素很可能提供对一种全新类型视网膜的洞察 和大脑钙传感器。对回收的兴趣进一步提高了 发现它是癌症相关性视网膜病变的抗原，以及 原发肿瘤引起的自身免疫性视网膜退行性疾病 远处的组织。

The overall goal of this research is to elucidate the molecular basis of visual excitation and adaptation in retinal rod cells. The focus will be on how calcium signals are read and processed in visual recovery and adaptation. The following enzymatic, spectroscopic, and electrophysiological studies of rod outer segment (ROS) proteins will be carried out: (1) The molecular mechanism of action of recoverin as a calcium sensor in vision will be defined by functional reconstitution of calcium-sensitive guanylate cyclase activity. Proteins in addition to recoverin and guanylate cyclase that are essential for conferring calcium- sensitive activation will be purified and characterized. In particular, the potential participation of the alpha subunit of transducin bearing GDP (T-alpha-GDP) will be assessed. (2) Does recoverin also control the activity of the cGMP phosphodiesterase (PDB), as does S-modulin in frog ROS? If so, the site of action of recoverin - whether it acts directly on photoexcited rhodopsin, rhodopsin kinase, transducin, or another component - will be delineated. (3) Retinal recoverin is acylated at its amino- terminus by myristic acid (14:0) or a related acyl group (14:1, 14:2, 12:0). Recombinant recoverins containing each of these acyl groups will be prepared. The effect of the attached acyl group on the calcium-induced insertion of recoverin into membranes will be measured. The geometry of membrane-inserted recoverin will be delineated by fluorescence energy transfer spectroscopy. (4) Proteins that bind to recoverin in a calcium- specific manner will be identified by affinity chromatography on recoverin-agarose, covalent crosslinking, and fluorescence emission anisotropy spectroscopy. (5) Site-specific mutants of recoverin will be produced to ascertain the functional significance of different regions. Of especial interest are the calcium-binding sites of EF hands 2 and 3, the hydrophobic crevice between EF hands 1 and 2, the linker between EF hands 3 and 4, and the highly charged C-terminal tail. (6) Electrophysiological studies of the effects of native and mutant recoverins and of T-alpha-GDP on membrane currents of truncated rods will be carried out to complement biochemical and spectroscopic studies. Studies of the mode of action of recoverin are likely to provide insight into a whole new class of retinal and brain calcium sensors. Interest in recoverin is further heightened by the finding that it is the antigen in cancer-associated retinopathy, an autoimmune retinal degenerative disease caused by a primary tumor in a distant tissue.