CNG Channel Gating Movements Monitored via Fluorescene Quenching

通过荧光淬灭监测 CNG 通道选通运动

• 批准号: 7485378
• 负责人: MICHAEL C PULJUNG
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7485378
• 关键词: Address; Affinity; Agonist; Binding; Biological Assay; Biology; Blindness; Brain; C-terminal; Cations; Circular Dichroism; Crystallography; Cyclic Nucleotides; DNA Sequence Rearrangement; Data; Disease; Eye; Fluorescence; Fluorometry; Genes; Goals; Guanine Nucleotide Exchange Factors; Helix (Snails); Human; Individual; Ion Channel; Ion Channel Gating; Ion Channel Protein; Label; Ligand Binding; Ligands; Measurement; Measures; Metal Binding Site; Modeling; Molecular; Monitor; Motion; Movement; Mutation; Nature; Operative Surgical Procedures; Phosphotransferases; Phototransduction; Physiology; Positioning Attribute; Protein Family; Proteins; Public Health; Relative (related person); Resolution; Rest; Retinal Degeneration; Retinitis Pigmentosa; Signal Transduction; Solutions; Structure; Testing; Tryptophan; Xenopus oocyte; achromatopsia; base; cadmium ion; cell growth regulation; cyclic-nucleotide gated ion channels; ear helix; fluorophore; insight; patch clamp; research study; structural biology; time use; transcription factor; visual information

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.

描述(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协同结合到保守的环核苷酸结合域(CNBD)调节的变构蛋白。CNG通道产生主要的电信号，负责将视觉和嗅觉信息传递到大脑。CNG通道的人类突变会导致各种疾病，包括完全性色盲或色盲以及视网膜色素变性，从而导致视网膜退化和失明。用X射线结晶学测定了HCN_2的C-末端结构域的配体结合结构，HCN_2是一个密切相关的离子通道，其中包括一个CNBD。这个结构域由一个八链卷曲和两个螺旋(B和C)组成，在C螺旋和卷曲之间的口袋中有环核苷酸结合。未连接的CNBD的结构和伴随配体结合和通道门控的运动的细节尚未确定。这项拟议研究的目标是通过对溶液中和完整通道中的CNBD进行荧光测量来监测这种构象变化。连接到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关于CNBD构象变化的性质的具体问题。在激动剂结合和通道门控时，C螺旋是如何相对于滚动重新定向的？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CNG通道的电流测量同时进行，这使得结构变化可以直接与功能蛋白质状态相关，这对于其他类型的蛋白质来说是不可能的。因此，所提出的研究不仅将是关于离子通道门控机制的信息，而且它们还将提供对结构相关分子的运动的洞察，包括某些激酶、转录因子和鸟嘌呤核苷酸交换因子。公共卫生相关性这项研究的目标是了解在一个被称为环核苷酸门控离子通道的蛋白质家族中发生的动态结构重排。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会导致失明。使用结构和功能分析相结合的方法，将确定这些通道的正常分子运动，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错误调节。