MECHANISM OF ION CHANNEL MODULATION BY LIGANDS

描述（由申请人提供）：环核苷酸调节通道在无数生理过程中发挥关键作用，包括视觉和嗅觉信号转导以及心脏和大脑中的起搏器活动。这些通道是功能性四聚体，并且环核苷酸（cAMP和cGMP）与通道的专门的保守区域的结合调节通道的打开/关闭（称为门控）平衡以及随后通道在信号级联中的作用的完成。这些通道的高分辨率结构和配体介导的通道激活的精确机制都尚不清楚。本提案的总体目标是了解环核苷酸调节离子通道的机制。为了实现这一目标，我们将利用这些通道的原核同源物MloK 1。脯氨酸通道本身的结构和生物化学研究，而结构的结论，真核生物的通道仅限于推理的基础上的功能数据。我们的第一个主要目标是确定环核苷酸结合和通道激活之间的联系。配体门控通道从第一配体的结合到通道激活经历一系列构象步骤，但分子细节是未知的。使用直接配体结合（首次使用这种技术的环核苷酸门控通道）和功能通量测定，我们将量化这一过程，以确定导致通道激活的步骤。我们的第二个主要目的是确定分子决定因素的通道响应配体和配体之间的选择性。为了确定配体选择性的分子决定因素，我们将破坏环核苷酸结合结构域与突变的残基已知影响配体的效力和选择性在真核细胞通道。然后，我们将评估突变的通道直接结合环核苷酸的能力和它们被环核苷酸调节的能力，并根据通道激活的模型量化这些影响。我们还将使用模块化的方法，在该方法中，我们将构建具有“混合和匹配”结构域的嵌合通道，以便为每个模块分配特定功能。这些实验将鉴定在配体结合和通道激活中起关键作用的单个残基以及蛋白质模块。我们的第三个主要目标是确定环核苷酸参与通道调节的结构元件。使用X射线晶体学，我们将确定MloK 1的结构和由稳定核心KcsA通道组成的功能通道嵌合体的结构，我们已经向该通道中添加了MloK 1的环核苷酸结合结构域。直接观察配体结合和孔结构域之间的相互作用将为门控机制提供独特的见解。配体和非配体通道结构之间的差异将提供深入了解配体门控的机制。嵌合通道结构代表了研究通道结构域的一种新方法。

DESCRIPTION (provided by applicant): Cyclic nucleotide-modulated channels play crucial roles in a myriad of physiological processes including visual and olfactory signal transduction and the pacemaker activity in the heart and brain. These channels are functional tetramers and the binding of cyclic nucleotides (cAMP and cGMP) to a specialized, conserved region of the channel modulates the opening/closing (called gating) equilibrium of the channel and the subsequent completion of the channel's role in the signaling cascade. Both the high resolution structure of these channels and the precise mechanism of ligand-mediated channel activation are unknown. The overall objective of this proposal is to understand the mechanism of ion channel modulation by cyclic nucleotides. To accomplish this goal we will utilize a prokaryotic homologue of these channels, MloK1. Prokaryotic channels lend themselves to structural and biochemical studies, while structural conclusions about eukaryotic channels are limited to inferences based on functional data. Our first major aim is to identify the linkage between cyclic nucleotide binding and channel activation. Ligand-gated channels go through a series of conformational steps from the binding of the first ligand to channel activation but the molecular details are unknown. Using direct ligand binding (the first use of this technique for cyclic nucleotide-gated channels) and functional flux assays we will quantify this process to identify the steps that lead to channel activation. Our second major aim is to identify the molecular determinants of channel response to ligands and selectivity among ligands. In order to identify the molecular determinants of ligand selectivity, we will disrupt the cyclic nucleotide binding domain with mutations of residues known to affect ligand potency and selectivity in eukaryotic channels. We will then assess the capacity of the mutated channels to directly bind cyclic nucleotides and their ability to be modulated by cyclic nucleotides and quantify these effects in terms of a model for channel activation. We will also use a modular approach in which we will construct chimeric channels with "mixed and matched" domains in order to assign specific functions to each module. These experiments will identify individual residues as well as protein modules that play key roles in ligand binding and channel activation. Our third major aim is to determine the structural elements involved in channel modulation by cyclic nucleotides. Using X-ray crystallography, we will determine the structure of MloK1 and that of a functional channel chimera made of a stable core, the KcsA channel, to which we have added the cyclic nucleotide binding domain of MloK1. Direct observation of the interactions between the ligand binding and the pore domains will provide unique insights into the gating mechanism. Differences between liganded and unliganded channel structures will provide insight into the mechanism of ligand gating. The chimeric channel structures represent a novel approach in studying channel structural domains.