Dissecting the mechanism of ligand regulation in cyclic nucleotide gated channels at the single molecule level.

细胞通过细胞膜运输离子的能力是最基本的生物过程之一，并允许细胞与环境相互作用。离子的运输是由称为离子通道的蛋白质实现的，它为离子提供了穿过原本不透膜的细胞膜的途径。离子通道的开启和关闭(门控)响应于其他小分子(配体)的结合是许多信号转导途径中的关键步骤。环核苷酸门控(CNG)离子通道控制着同时允许视觉和嗅觉的信号机制。与这个离子通道家族的其他成员一样，CNG通道是由四个单独的亚基组装而成，每个亚基的一部分对通道结构有贡献。每个亚基都包含一个配体结合位点，与蛋白质这些部分的结合被认为会引起结构变化，进而导致离子通道的打开/关闭。这个配基结合、转导和通道选通的过程是理解离子通道中配基选通的动态分子机制的核心。更好地了解CNG中的配体调控将有助于我们设计方法来操纵这些重要的信号机制。我们将开发能够在单个分子水平上同时测量配体结合和离子通道门控的仪器。通过在单分子水平上观察配体结合和转导，我们将能够研究这一信号转导过程中的每一步；既观察配体结合事件，又观察离子通道的后续反应。我们将使用这种方法来研究嵌合的环核苷酸门控钾通道KCNG中配体调节的分子机制。

The ability of a cell to transport ions across the cell membrane is one of the most fundamental biological processes and allows a cell to interact with its environment. Transport of ions is achieved by proteins called ion channels, that provide pathways for ions to cross the otherwise impermeable cell membrane. The opening and closing (gating) of ion channels in response to the binding of other small molecules (ligands) is a critical step in many signal transduction pathways. The cyclic-nucleotide gated (CNG) ion channel governs the signalling mechanisms that permit both sight and smell. Like other members of this family of ion channels, CNG channels are formed by the assembly of four individual subunits, with a portion of each subunit contributing to the channel structure. Each subunit contains a ligand-binding site, and binding to these portions of the protein is thought to cause an structural change that is transduced into opening/closure of the ion channel. This process of ligand binding, transduction, and channel gating is central to understanding the dynamic molecular mechanism of ligand gating in ion channels. A better understanding of ligand regulation in CNG would help us in designing methods to manipulate these important signalling mechaims. We will develop apparatus capable of simultaneous measurements of ligand binding and ion channel gating at the level of individual molecules. By observing ligand binding and transduction at the single-molecule level we will be able to study each step in this signal transduction process; observing both the ligand binding event and the subsequent response of the ion channel. We will use this approach to address the molecular mechanism of ligand regulation in a chimeric cyclic-nucleotide gated potassium channel, KCNG.