THE ACTIVATION GATE OF CYCLIC NUCLEOTIDE-GATED CHANNELS

描述（由申请人提供）：环核苷酸门控（CNG）通道在光感受器神经元中发挥重要作用，介导由光子吸收触发的信号转导级联的最后一步。它们是通过结合环核苷酸打开的Ca 2+可渗透的非选择性阳离子通道。CNG通道基因的突变导致视觉系统中的许多疾病，包括视网膜色素变性（视网膜变性）和全色盲（全色盲）。CNG通道是含有P环的离子通道大家族的成员，其具有相当大的共同结构和功能相似性。所提出的实验的长期目标是了解通道蛋白中的构象重排如何导致CNG通道孔的打开。虽然最近的细菌P-环通道的晶体结构揭示了闭孔和开孔构象，但关于通道门控的分子机制的许多基本问题仍然没有答案。什么样的孔隙结构构成激活门？引发离子渗透的结构运动是什么？为了解决CNG通道中的这些问题和相关问题，该赠款将特别利用我们最近开发的荧光技术（称为膜片钳荧光测定法或PCF），该技术基于孤立膜补丁中通道的位点特异性荧光标记。PCF将用于在真实的时间内同时记录来自相同通道群体的荧光和电流信号，并将通道蛋白的结构变化与其门控效应直接关联。PCF将与荧光猝灭和荧光共振能量转移（FRET）相结合，以研究CNG通道激活过程中通道孔结构以及结构中的重排。这些实验的发现将促进我们对生理和疾病状态下CNG通道门控的分子基础的理解。

DESCRIPTION (provided by applicant): Cyclic nucleotide-gated (CNG) channels play a fundamental role in photoreceptor neurons where they mediate the final step of the signal transduction cascade triggered by absorption of photons. They are Ca2+ permeable, non-selective cation channels that are opened by binding of cyclic nucleotides. Mutations in CNG channel genes are responsible for numerous diseases in the visual system including retinitis pigmentosa (retinal degeneration) and achromatopsia (total color blindness). CNG channels are members of a large family of P-loop containing ion channels that shares considerable common structural and functional similarities. The long-term goal of the proposed experiments is to understand how conformational rearrangements in the channel protein lead to the opening of the CNG channel pore. While recent crystal structures of bacteria P-loop containing channels revealed both the closed and the open pore conformations, many fundamental questions regarding the molecular mechanism of channel gating remain unanswered. What pore structure constitutes the activation gate(s)? What are the structural movements that initiate ion permeation? To address these and related questions in CNG channels, the grant will take particular advantage of our recently developed fluorescence technique (termed Patch-Clamp Fluorometry, or PCF) that is based on site-specific fluorescence labeling of channels in isolated membrane patches. PCF will be used to simultaneously record fluorescence and current signals from the same population of channels in real time, and to directly correlate structural changes in the channel protein to their gating effects. PCF will be combined with fluorescence quenching and fluorescence resonance energy transfer (FRET) to investigate the channel pore structure as well as rearrangements in the structure during CNG channel activation. Findings from these experiments will advance our understanding of the molecular basis for CNG channel gating under physiological as well as disease states.