ISOLATION OF THE LIGHT-MODULATED CHANNEL OF RETINAL RODS

通过一系列的酶反应，脊椎动物的光感受器 将吸收一个光子所产生的化学能转化为 视紫红质在Plamsa膜上转化为电信号。这个 与视觉有关的光化学和生物化学序列相当好 定义了我们对光感受器上的电事件的理解 膜是不发育的。该项目旨在提供分子 对感光细胞外段通透性快速变化的洞察 对光的响应以及对膜结构的洞察 对这些渗透率的变化负责。 我们提出了一种多学科的方法，使用熟悉的生化和 适用于研究这种新型传输的生物物理技术 机制。分离的牛外节膜，剥离 磷酸二酯酶将被融合到平面脂质双层中，以定义 环状GMP门控的生物物理特性。双层电流将是 与cGMP对蛙棒外膜内向外贴片影响的比较 细分市场。电生理技术将定义cGMP 浓度-电导关系、单通道性质、离子 这种新型电导的选择性和离子-离子相互作用。水泡 适用于22nA磁导率的测量将由ROS形成 膜用于测定cGMP依赖的阳离子通透性 直接去吧。将使用内流研究来量化45Ca和22Na 生理条件下的内流并确定哪些因素会影响 选择性。 尽管cGMP与其受体结合有很高的解离常数 30um，~3H-cGMP可用于确定 反应是因为高位点密度。结合协作性和 受体密度将被估计。分子的荧光偏振 (硫代荧光素)-cGMP将用于确定 洗涤剂的增溶组分及最佳增溶条件 将会被定义。将使用标准的生化技术来分离 CGMP受体。提纯的组分将被重组为脂类 并检测cGMP依赖的通透性变化。

Through a cascade of enzymatic reactions, vertebrate photoreceptors transduce the chemical energy produced by the absorption of a photon by rhodopsin into an electrical signal at the plamsa membrane. The photochemical and biochemical sequences involved in vision are fairly well defined but our understanding of the electrical events on the photoreceptor membrane is rudimentary. This project is designed to provide molecular insight into the rapid changes in photoreceptor outer segment permeability in response to light as well as insight about the membrane structure responsible for these permeability changes. We propose a multidisciplinary approach using familar biochemical and biophysical techniques adapted to the study of this novel transport mechanism. Isolated bovine outer segment membranes, stripped of phosphodiesterase, will be fused into planar lipid bilayers to define the biophysical properties of cyclic GMP gating. Bilayer currents will be compared with cGMP effects on inside-out patches from frog rod outer segments. Electrophysiological techniques will define the cGMP concentration-conductance relationship, single channel properties, ion selectivity, and ion-ion interactions of this novel conductance. Vesicles suitable for 22Na permeability measurements will be formed from ROS membranes in order to assay the cGMP-dependent cation permeability directly. Influx studies will be used to quantitate the 45Ca and 22Na influx under physiological conditions and determine what factors influence the selectivity. Despite the high dissociation constant for cGMP binding to the receptor of 30 uM, 3H-cGMP can be used to define the chemical equilibria of the reaction because of the high site density. The binding cooperativity and receptor density will be estimated. Fluorescence polarization of (thio-fluorescein)-cGMP will be used to determine the binding in solubilized fractions and optimal conditions for detergent solubilization will be defined. Standard biochemical techniques will be used to isolate the cGMP receptor. Purified fractions will be reconstituted into lipid vesicles and assayed for cGMP-dependent permeability changes.