ISOLATION OF THE LIGHT-MODULATED CHANNEL OF RETINAL RODS

视网膜杆光调制通道的隔离

• 批准号: 3263136
• 负责人: JACQUELINE C TANAKA
• 金额: $ 18.57万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3263136
• 关键词: Anura; bioenergetics; biological signal transduction; biophysics; calcium; calcium channel blockers; cations; chemical binding; cyclic GMP; electrophysiology; fluorescence; fresh water environment; ionophores; ligands; light adaptations; membrane channels; membrane permeability; membrane proteins; nucleotide analog; phosphorylation; photochemistry; sodium; visual photoreceptor; voltage /patch clamp

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.

从光感受器外部转导生化信息 片段光盘的质膜电导变化是主要的 视觉处理中的事件。我们现在了解到CGMP是 终端，在生化级联中的可扩散发射器打开 质膜离子通道。 多个CGMP分子合作结合 到达通道以快速开关膜电导。 给出 这个数字类型开关，感光器如何产生类似物 从总黑暗中的如此广泛的条件下的类型响应 阳光明媚。 其他人最近进行的实验表明CA播放 在适应中的重要作用。 我们的研究重点是回答 关于分子水平的问题有关核苷酸激活的问题 通道和CA在通道功能中的作用。 我们的具体目的是1）解决 通道上的核苷酸结合位点，2）发展荧光或 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CA与 Na/ca竞争的通道和特性，4） 为了检查营地对电流的代谢调节， 质子和磷酸化5）确定 使用有机阳离子的eRies通道。 大多数数据将是 从从电压钳等离子体记录的离子电流获得 CGMP或衍生物已暴露于 细胞质面。 配体激活的动力学特征将是 从宏观静脉注射的组合获得，电流噪声 在电压夹下，瞬时IV记录和电压跳。 这 单通道电导将根据当前噪声和 低浓度核苷酸的单个通道记录。 为了确定二价阳离子如何渗透并阻止此通道我们 将使用几种经典的电生理方法。 我们CA 在我们改变浴室中Na/Ca的派系时，记录电流。 到 消除门控效应，我们还可以在我们的时候测量瞬时IV 增加[CA}。 目前，我们将CA视为一个药水通道 阻滞剂。 我们想知道的部分是频道是否 选择性随代谢状态的变化而变化，例如 磷酸化或质子化将在完整的感光体中进行 与W. Cobbs博士合作。 最后我们将探究物理 使用一系列有机阳离子开始的通道的尺寸 使用nh4+，一个非常渗透的离子，并使用茶，tma增加尺寸 和胆碱。