MOLECULAR PROPERTIES OF A CGMP-ACTIVATED CHANNEL

CGMP 激活通道的分子特性

• 批准号: 3288139
• 负责人: STANLEY M GOLDIN
• 金额: $ 22.71万
• 依托单位: CAMBRIDGE NEUROSCIENCE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-06-01 至 1992-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288139
• 关键词: acetylcholine; axon; brain; calcium channel; calcium transporting ATPase; cations; cow; cyclic GMP; density gradient ultracentrifugation; diltiazem; high performance liquid chromatography; isomer; membrane potentials; membrane proteins; membrane reconstitution /synthesis; protein structure; radiotracer; synaptosomes; transport proteins; visual photoreceptor

It has recently been observed that guanosine 3':5' cyclic monophosphate ("cGMP") activates cation channels in the outer segments of vertebrate rod photoreceptor cells. Our laboratory has developed a procedure for purification of discs from bovine rod outer segments, and demonstrated that such a channel is associated with discs. When activated by cGMP, the channel can rapidly discharge a pool of Ca++ that is actively accumulated by a disc- associated, ATP-dependent Ca++ pump. Both cGMP and Ca++ are capable of modulating the membrane potential of rod outer segment plasma membranes, and light initiaties events that dramatically alter cGMP metabolism; this suggests that the cGMP-activated channel of discs may be an important link in the short and/or long term regulation of photoreceptor electrical activity. Our goals are to further characterize, reconstitute and purify this cGMP-activated channel. To quantitatively characterize the activation, pharmacology, and conductance properties of the channel, we will employ-- and further refine-- new approaches for rapid kinetic measurement of channel activity. The propsed rapaid kinetic techniques may be of general applicability for characterization of ion channels residing in organelles that are too small for application of electrophysiological methods such as patch clamping. To purify the disc channel, we will employ a novel approach this laboratroy has developed--"transport- specific fractionation"-- together with other methods for membrane protein purification. Transport-specific fractionation uses reconstitution of membrane transport proteins into liposomes as a physical tool for transport protein purification in functionally active form. Identification of the associated agoinst and antagonist binding sites will be an additional means of monitoring the channel's reconstitution and purification. Previously documented regulatory effects of cGMP In other systems appear to be phosphokinases- mediated; in contrast, there are strong indications that both the disc-associated channel-- and a similar or identical one in the plasma membrane of rod other segments-- are directly activated by cGMP. Reconstitution and purification of the channel should enable use to rigorously test this important new hypothesis.

最近观察到鸟苷 3':5' 环状 单磷酸盐（“cGMP”）激活外部的阳离子通道 脊椎动物视杆感光细胞的片段。 我们的实验室 开发了一种从牛中纯化椎间盘的程序 杆外段，并证明了这样的通道是 与光盘相关。 当被 cGMP 激活时，该通道可以 快速排出由 a 积极积累的 Ca++ 池 椎间盘相关、ATP 依赖性 Ca++ 泵。 cGMP 和 Ca++能够调节杆的膜电位 外段质膜和光引发事件 显着改变 cGMP 代谢；这表明 cGMP 激活的椎间盘通道可能是 感光器电的短期和/或长期调节 活动。 我们的目标是进一步表征、重构和 净化这个 cGMP 激活的通道。 定量表征活性、药理学和 通道的电导特性，我们将采用 - 和 进一步完善——快速动力学测量的新方法 渠道活动。 所提出的快速动力学技术可能是 离子通道表征的一般适用性 细胞器太小而无法应用 电生理方法，例如膜片钳。 为了净化光盘通道，我们将采用一种新颖的方法 Laboratroy 开发了——“运输特定分馏”—— 与其他膜蛋白纯化方法一起使用。 运输特异性分级分离使用膜重构 将蛋白质运输到脂质体中作为运输的物理工具 以功能活性形式纯化蛋白质。 鉴定 相关的激动剂和拮抗剂结合位点将是 监控通道重建的附加手段和 纯化。 先前记录的 cGMP 在其他方面的监管影响 系统似乎是磷酸激酶介导的；相比之下，有 强烈表明与光盘相关的通道 - 和 其他杆的质膜中的相似或相同的 片段——由 cGMP 直接激活。 重组和 通道的净化应能够用于严格测试 这个重要的新假设。