GATING MECHANISMS OF RETINAL ROD cGMP ACTIVATED CHANNELS

描述(申请人提供)：环核苷酸门控(CNG)离子通道对光感受器中的光和嗅觉感受器中的气味产生初级电反应。它们是非选择性的阳离子通道，通过环核苷酸与通道的直接结合而打开。这些通道因其在信号转导中的作用而高度专业化。拟议中的实验的长期目标是了解这些专业化背后的分子机制。在过去的几年中，这些通道的变构激活和调节涉及到通道的多个结构域之间的动态相互作用：环核苷酸结合结构域(CNBD)、孔、连接CNBD和孔的C-连接结构域以及氨基末端区域。然而，一些基本的问题仍然存在：与通道开放相关的CNBD和C-连接器域的结构重排是什么？在选通过程中，亚基相互作用是如何变化的？与钙-钙调蛋白调节通道相关的结构重排是什么？为了解决这些和相关的问题，这笔赠款将特别利用两项令人振奋的新进展：1)我们解决了密切相关的HCN通道的细胞内配体结合和门控区域的X射线晶体结构，以及2)我们开发了CNG通道细胞内区域的定点荧光标记和无细胞膜片中的荧光记录技术(称为膜片钳荧光法，PCF)。HCN2通道的结构表明，C-接头形成了一个新的通道四聚结构域，位于CNBD和孔道之间。此外，生化和电生理实验表明，这些亚基的相互作用是动态的。这些实验将使用突变分析和PCF来研究CNG CNBD、C-接头和与通道激活和调制相关的氨基末端区域的重排。这一结果将为深入了解CNG通道的正常行为及其在疾病状态下的故障机制提供依据。

DESCRIPTION (provided by applicant): Cyclic nucleotide-gated (CNG) ion channels generate the primary electrical response to light in photoreceptors and to odorant in olfactory receptors. They are nonselective cation channels that are opened by the direct binding of cyclic nucleotides to the channel. The channels are highly specialized for their role in signal transduction. The long-term goal of the proposed experiments is to understand the molecular mechanisms that underlie these specializations. In the last several years, the allosteric activation and modulation of these channels has been shown to involve dynamic interactions between multiple domains of the channel: the cyclic nucleotide-binding domain (CNBD), the pore, the C-linker domain connecting the CNBD to the pore, and the amino-terminal region. However a number of fundamental questions still remain: What are the structural rearrangements in the CNBD and C-linker domains that are associated with channel opening? How do the subunit interactions change during gating? What are the structural rearrangements associated with Ca2+-calmodulin modulation of the channel? To address these and related questions, this grant will take particular advantage of two exciting new developments: 1) our solution of the x-ray crystal structure of the intracellular ligand binding and gating domains of the closely related HCN2 channel, and 2) our development of techniques for site-specific fluorescent labeling of the intracellular domain of CNG channels and recording fluorescence in cell-free membrane patches (termed patch-clamp fluorometry, PCF). The structure of the HCN2 channel reveals that the C-linker forms a novel tetramerization domain of the channel, situated between the CNBD and the pore. Furthermore, biochemical and electrophysiological experiments suggest that these subunit interactions are dynamic. These experiments will use mutational analysis and PCF to investigate the rearrangements in the CNG CNBD, C-linker, and amino-terminal region associated with channel activation and modulation. The results will provide insights into the mechanisms for the normal behavior of CNG channels and their malfunction in disease states.