GATING MECHANISMS OF RETINAL ROD cGMP ACTIVATED CHANNELS

描述（由申请人提供）：环核苷酸门控（CNG）离子通道在光感受器中产生对光的主要电反应，在嗅觉感受器中产生对气味的主要电反应。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。这些通道对于它们在信号转导中的作用是高度特化的。拟议实验的长期目标是了解这些专业化的分子机制。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，连接CNBD到孔的C-接头结构域，和氨基末端区域。然而，一些基本的问题仍然存在：什么是CNBD和C-接头结构域的结构重排与通道开放？门控期间亚基相互作用如何变化？什么是结构重排与Ca 2 +-钙调素调节通道？为了解决这些问题和相关问题，这笔赠款将特别利用两个令人兴奋的新发展：1）我们对密切相关的HCN 2通道的细胞内配体结合和门控结构域的X射线晶体结构的解决方案，2）我们开发了CNG通道胞内区域的位点特异性荧光标记技术和记录无细胞膜补丁中的荧光（称为膜片钳荧光测定法，PCF）。HCN 2通道的结构表明，C-接头形成了一个新的四聚化结构域的通道，位于CNBD和孔之间。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来研究与通道激活和调节相关的CNG CNBD、C-接头和氨基末端区域中的重排。这些结果将为CNG通道的正常行为及其在疾病状态下的功能障碍提供深入了解。

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.