ISOLATION OF THE LIGHT-MODULATED CHANNEL OF RETINAL RODS

光感受器外部生化信息的传递 盘段的电导变化主要是在质膜上 事件发生在视觉处理中。我们现在了解到cGMP的功能是 终端，生化级联中的扩散发射器，以打开一个 质膜离子通道。多个cGMP分子协同结合 该通道可快速开启膜电导。鉴于此， 数字式开关，光感受器如何产生模拟型开关 对如此广泛的条件做出反应，从完全黑暗到明亮 阳光。最近其他人的实验表明，钙在人体内 在适应中的作用。我们的研究集中在回答问题上 关于核苷酸激活通道的分子水平及其作用 钙离子在通道功能中的作用。 我们的具体目标是：1)解析分子的结构 通道上的核苷酸结合部位，2)形成荧光或 放射性标记的cGMP类似物可用于定量合作 结合等温线3)了解钙与通道的相互作用和 Na/Ca竞争渗透的性质，4)考察可能 CAMP、质子和磷酸化对电流的代谢调节 5)使用一系列有机材料确定航道的物理尺寸 阳离子。大部分数据将从记录的离子流中获得 来自电压钳制的质膜切除的补片，其中cGMP或 衍生物已经暴露在细胞质表面。其动力学特征 配体活化将从宏观IV的组合中获得 录音、电压钳位下的电流噪声、瞬时IV录音 然后电压就会跳跃。单通道电导的估计式为 低浓度下的电流噪声和单个通道记录 核苷酸。 为了确定二价阳离子如何渗透和阻断这一通道，我们将 使用几种经典的电生理学方法。我们可以录制 电流，因为我们改变了浴液中Na/Ca的分数。消除门控的步骤 当我们增加[Ca]时，我们也可以测量瞬时IV。 目前，我们认为钙是一种有意的通道阻滞剂。我们想要的是 要知道，在一定程度上，频道选择性是否随着 代谢状态，如磷酸化或质子化，将在 完整的光感受器，与W.Cobbs博士合作。最后我们会 用一系列有机的方法探测河道的物理尺寸 从NH4+开始的阳离子，一种非常有意义的离子，并增加了尺寸 使用茶、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 can 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.