MOLECULAR PROPERTIES OF A CGMP-ACTIVATED CA CHANNEL

最近观察到鸟苷3 '：5'环 一磷酸盐（“cGMP”）激活外膜中的阳离子通道。 脊椎动物视杆细胞的节。 本实验室 已经开发了一种从牛中纯化圆盘的方法， 杆外节，并证明，这样的通道是 与光盘有关。 当被cGMP激活时，该通道可以 快速释放由细胞主动积累的Ca++池， 椎间盘相关的ATP依赖性Ca++泵。 cGMP和 Ca ~（++）可调节视杆细胞膜电位 外节质膜，以及光初始化事件， 显著改变cGMP代谢;这表明， cGMP激活的椎间盘通道可能是神经元凋亡的重要环节。 光感受器电学短期和/或长期调节 活动 我们的目标是进一步描述、重组和 纯化这个cGMP激活的通道。 为了定量表征活化、药理学和 通道的电导特性，我们将采用-和 进一步完善-快速动态测量 渠道活动。 提出的辐射动力学技术可能是 表征离子通道的一般适用性 细胞器太小， 电生理学方法，如膜片钳。 为了净化椎间盘通道，我们将采用一种新的方法， 实验室已经开发出了--“运输特定分馏”-- 以及用于膜蛋白纯化的其它方法。 转运特异性分馏使用膜的重构 将蛋白质转运到脂质体中作为转运的物理工具 功能活性形式的蛋白纯化。 鉴定 相关的激动剂和拮抗剂结合位点将是 监测通道重建的附加手段， 洁净. 以前记录的cGMP在其他领域的监管作用 系统似乎是磷酸激酶介导的;相反， 强烈表明，与光盘相关的通道-- 在杆细胞膜上有一个相似或相同的 片段直接被cGMP激活。 复溶和 通道的净化应能使使用者严格地测试 这个重要的新假设

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.