CNG Channel Gating Movements Monitored via Fluorescene Quenching

描述（由申请人提供）：环核苷酸门控（CNG）离子通道是由细胞质环核苷酸与保守环核苷酸结合域（CNBD）协同结合调节的变构蛋白。CNG通道产生主要电信号，负责将视觉和嗅觉信息转导到大脑。人类CNG通道的突变导致各种疾病，包括完全性色盲或色盲和视网膜色素变性，导致视网膜变性和失明。HCN2是一种密切相关的离子通道，其c端结构域的配体结合结构，包括CNBD，已被x射线晶体学确定。这个结构域是由一个八链螺旋和两个螺旋（B和C）组成的，环状核苷酸结合在C螺旋和螺旋之间的口袋里。未配体CNBD的结构以及伴随配体结合和通道门控的运动细节尚未确定。该研究的目的是利用荧光测量分离的CNBD在溶液和完整的通道中监测这种构象变化。在通道上不同位置的荧光团和猝灭剂之间的距离依赖猝灭将解决有关CNBD构象变化性质的具体问题。在激动剂结合和通道门控时，C螺旋是如何相对于卷筒重新定向的？在没有激动剂的情况下，C螺旋是否保持其二级结构？荧光测量可以与来自CNG通道的电流测量同时进行，这允许结构变化与功能蛋白质状态直接相关，这是其他类型蛋白质无法实现的分辨率。因此，拟议的研究不仅将提供有关离子通道门控机制的信息，而且还将提供对结构相关分子运动的洞察，包括某些激酶，转录因子和鸟嘌呤核苷酸交换因子。本研究的目的是了解发生在被称为环核苷酸门控离子通道的蛋白质家族中的动态结构重排。这些蛋白质是将视觉信息从眼睛传递到大脑所必需的，编码这些通道的基因突变导致失明。通过结构和功能分析的结合，这些通道的正常分子运动将被确定，这将有助于理解健康个体中细胞信号对它们的调节，以及它们在疾病中的失调。

DESCRIPTION (provided by applicant): Cyclic nucleotide-gated (CNG) ion channels are allosteric proteins regulated by the cooperative binding of cytoplasmic cyclic nucleotides to a conserved cyclic nucleotide binding domain (CNBD). CNG channels generate the primary electrical signal responsible for transduction of visual and olfactory information to the brain. Human mutations in CNG channels cause various diseases including complete achromatopsia or colorblindness and retinitis pigmentosa, which leads to retinal degeneration and blindness. The ligand-bound structure of the C-terminal domain of HCN2, a closely related ion channel, which includes a CNBD, has been determined by x-ray crystallography. This domain is formed by an eight-stranded ¿ roll followed by two helices (B and C) with cyclic nucleotide binding in a pocket between the C helix and the ¿ roll. The structure of the unliganded CNBD and the details of the movements accompanying ligand binding and channel gating have not been determined. The goal of the proposed study is to monitor this conformational change using fluorescence measurements of isolated CNBD's in solution and intact channels. Distance-dependent quenching between fluorophores and quenchers attached to various positions on the channel will address specific questions about the nature of the CNBD conformational change. How does the C helix reorient relative to the ¿ roll upon agonist binding and channel gating? Does the C helix maintain its secondary structure in the absence of agonist? Fluorescence measurements can be performed simultaneously with current measurements from CNG channels, which allows for structural changes to be correlated directly with a functional protein state, a resolution not possible for other types of proteins. Therefore, not only will the proposed studies be informative with respect to ion channel gating mechanisms, but they will also provide insight into the movements of structurally-related molecules, including certain kinases, transcription factors and guanine nucleotide exchange factors. PUBLIC HEALTH RELEVANCE The goal of this study is to understand the dynamic structural rearrangements that occur in a family of proteins known as cyclic nucleotide-gated ion channels. These proteins are necessary to relay visual information from the eye to the brain, and mutations in the genes encoding these channels result in blindness. Using a combination of structural and functional assays, the normal, molecular motions of these channels will be determined, which will be instrumental in understanding their regulation by cellular signals in healthy individuals, and their misregulation in disease.