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 以快速开启膜电导。vt.给出 这种数字类型的开关，光感受器如何产生模拟- 在从完全黑暗的如此广泛的条件下进行类型响应 为了灿烂的阳光。最近其他人的实验表明，钙在 在适应过程中起着重要作用。我们的研究重点是回答 在分子水平上关于核糖核酸激活的问题 通道及钙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 协同结合等温线3)理解钙与钙的相互作用 Na/Ca竞争通透的通道和特性，4) 为了研究cAMP对电流的可能代谢调节， 质子和磷酸化决定了分子的物理尺寸 使用一系列有机阳离子的通道。大多数数据将是 从电压钳制等离子体记录的离子电流获得 CGMP或其衍生物暴露于其中的膜切除斑块 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 在电压钳下，瞬时IV记录和电压跳跃。这个 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 当我们改变镀液中Na/Ca的比例时，记录电流。至 消除选通影响，我们还可以测量瞬时IV 增加[Ca]。目前，我们认为钙是一种预想的渠道 拦截者。我们想知道的是，在一定程度上， 选择性随着代谢状态的变化而变化，例如 磷酸化或质子化将在完整的光感受器中进行 与W.Cobbs博士合作。最后我们将探索物理上的 使用一系列有机阳离子开始的通道尺寸 对于NH4+，一种非常重要的离子，并使用TEA增加尺寸，TMA 还有胆碱。

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.