ISOLATION OF THE LIGHT-MODULATED CHANNEL OF RETINAL RODS

光感受器外膜生化信息的传递 节盘对质膜电导的改变是主要的 视觉处理中的事件。我们现在知道cGMP的功能是 终端，在生化级联扩散递质打开一个 质膜离子通道 多个cGMP分子协同结合 迅速开启膜电导。 给定 这种数字式开关，感光器如何产生模拟信号， 在如此广泛的条件下，从完全黑暗的类型反应 到明亮的阳光。 其他人最近的实验表明，钙起着 在适应中的重要作用。 我们的研究重点是回答 在分子水平上的问题有关的核苷酸激活的 通道和钙在通道功能中的作用。 我们的具体目标是：1）解决 通道上的核苷酸结合位点，2）产生荧光或 放射性标记的cGMP类似物，其可用于定量 协同结合等温线3）理解Ca与 Na/Ca竞争渗透的通道和性质; 4） 为了检查cAMP对电流的可能的代谢调节， 质子和磷酸化5）决定了 通道使用一系列的有机阳离子。 大部分数据将 从电压钳位等离子体记录的离子电流获得 膜切除贴片，其中cGMP或衍生物已暴露于 细胞质的脸。 配体活化的动力学特征将是 从宏观IV记录、电流噪声 欠压钳位、瞬时IV记录和电压跳变。 的 单通道电导将根据电流噪声和 在低浓度核苷酸下的单个通道记录。 为了确定二价阳离子是如何渗透并阻断该通道的， 将使用几种经典的电生理学方法。 WE CA 当我们改变浴中Na/Ca的比例时，记录电流。 到 消除门控效应，我们还可以测量瞬时IV，因为我们 增加[Ca]。 目前我们把Ca看作是一种渗透通道 修颜乳液 我们想知道的是，在某种程度上， 选择性随着代谢状态的变化而变化， 磷酸化或质子化将在完整的光感受器中进行， 与W博士合作科布斯 最后，我们将探讨物理 使用一系列有机阳离子的通道尺寸， 与NH 4+，一个非常渗透的离子，并增加大小使用TEA，TMA 和胆碱。

The transduction of biochemical information from the photoreceptor outer segment discs to conductance changes in the plasma membrane is a primary event in visual processing. We now understand that cGMP functions as the terminal, diffusible transmitter in the biochemical cascade to open a plasma membrane ion channel. Multiple cGMP molecules cooperatively bind to the channel to rapidly switch on the membrane conductance. Given this digital-type switch, how does a photoreceptor produce an analog- type response over such a wide range of conditions from total darkness to bright sunlight. Recent experiments by others suggest that Ca plays an important role in adaptation. Our research is focused on answering questions at the molecular level about the nucleotide activation of the channel and the role of Ca in channel function. Our specific aims are 1) to resolve the molecular architecture of the nucleotide binding site on the channel, 2) develop fluorescent or radiolabelled cGMP analogs which can be used to quantitate the cooperative binding isotherm 3) understand the interaction of Ca with the channel and the properties of Na/Ca competition for permeation, 4) to examine possible metabolic regulation of the currents by cAMP, protons, and phosphorylation 5) determine physical dimensions of the channel using a eries of organic cations. Most of the data will be obtained from ionic currents recorded from voltage-clamped plasma membrane excised patches where cGMP or a derivative has been exposed to the cytoplasmic face. Kinetic features of the ligand activation will be obtained from a combination of macroscopic IV recordings, current noise under voltage clamp, instantaneous IV recordings and voltage jumps. The single channel conductance will be estimated from current noise and from individual channel recordings at low concentrations of nucleotide. To determine how divalent cations permeate and block this channel we will use several classical electrophysiological approaches. WE ca record the current as we change the faction of Na/Ca in the bath. To eliminate gating effects we can also measure the instantaneous IV as we increase the [Ca}. At the present time we view Ca as a permeant channel blocker. What we want to know, in part, is whether the channel selectivity varies with changes in the metabolic state such as phosphorylation or protonation will be done in intact photoreceptors in collaboration with Dr. W. Cobbs. Finally we will probe the physical dimensions of the channel using a series of organic cations beginning with NH4+, a very permeant ion, and increasing the size using TEA, TMA and choline.