GATING MECHANISMS OF RETINAL ROD cGMP ACTIVATED CHANNELS

视网膜杆 cGMP 激活通道的门控机制

• 批准号: 7004525
• 负责人: William N Zagotta
• 金额: $ 29.61万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7004525
• 关键词: SDS polyacrylamide gel electrophoresis; Xenopus; allosteric site; alternatives to animals in research; binding proteins; binding sites; conformation; cyclic GMP; electrophysiology; fluorescence resonance energy transfer; fluorimetry; membrane channels; molecular cloning; phosphorylation; polymerase chain reaction; protein protein interaction; protein structure function; rod cell; single cell analysis; site directed mutagenesis; surface plasmon resonance; visual photoreceptor; visual phototransduction; voltage /patch clamp; voltage gated channel; yeast two hybrid system

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.

描述（由申请人提供）：环核苷酸门控（CNG）离子通道产生对光感受器中的光和嗅觉受体中的气味的主要电响应。它们是非选择性阳离子通道，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。所提出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已被证明涉及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、将 CNBD 连接到的 C 连接结构域。孔和氨基末端区域。然而，仍然存在一些基本问题：与通道开放相关的 CNBD 和 C-linker 结构域中的结构重排是什么？门控过程中亚基相互作用如何变化？与 Ca2+-钙调蛋白通道调节相关的结构重排是什么？为了解决这些和相关问题，这笔资助将特别利用两个令人兴奋的新进展：1）我们对密切相关的 HCN2 通道的细胞内配体结合和门控域的 X 射线晶体结构的解决方案，以及 2）我们的开发CNG 通道胞内结构域的位点特异性荧光标记和记录无细胞膜片中的荧光（称为膜片钳荧光测定法，PCF）的技术。 HCN2 通道的结构揭示了 C 连接体形成了通道的新型四聚化结构域，位于 CNBD 和孔之间。此外，生化和电生理学实验表明这些亚基相互作用是动态的。这些实验将使用突变分析和 PCF 来研究与通道激活和调节相关的 CNG CNBD、C 连接子和氨基末端区域的重排。研究结果将为了解 CNG 通道的正常行为及其在疾病状态下的功能障碍的机制提供见解。