Molecule Modulators:Voltage-Dependent Potassium (RMI)

分子调节剂：电压依赖性钾 (RMI)

• 批准号: 7058418
• 负责人: Ming Zhou
• 金额: $ 0.48万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2006-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7058418
• 关键词: NAD(H) phosphate; X ray crystallography; drug discovery /isolation; drug screening /evaluation; electrophysiology; enzyme substrate; high throughput technology; potassium channel; protein structure; protein structure function; recombinant proteins; small molecule; voltage gated channel; NAD(H) phosphate; X ray crystallography; drug discovery /isolation; drug screening /evaluation; electrophysiology; enzyme substrate; high throughput technology; potassium channel; protein structure; protein structure function; recombinant proteins; small molecule; voltage gated channel

DESCRIPTION (provided by applicant): Voltage-dependent potassium channels (Kv) are integral membrane proteins that, in response to membrane voltage changes, catalyze potassium ions to diffuse across the cell membrane. Kv channels regulate membrane excitability and are essential to many physiological processes, such as the rhythmic beating of heart, the communication between neurons, and the secretion of hormones. In a cell, Kv channels are always assembled with other proteins, and channel function is regulated by cellular environment through these associated proteins. The beta subunit (Kvbeta), a potential aldo-keto reductase (AKR), attaches to the intracellular side of the Kv1 family channels. They are ubiquitous in the brain and abundantly expressed in heart, and have been implicated in control of cell excitability and in sensing cellular redox state. The long-term goal of this research is to understand the role of Kvbeta in cell biology. We have recently identified two Kvbeta substrates and for the first time demonstrated that Kvbeta is a functional AKR. We also found that the substrates, when perfused to the intracellular side of the channel, modulate channel functions only when Kvbeta is co-expressed. These exciting new results led us to hypothesize that Kv1 channel functions are coupled to Kvbeta enzymatic activities. In this proposal we focus on screening for Kvbeta substrates and we propose the following two specific aims: Specific Aim 1. To identify small-molecule substrates of Kvbeta by a high throughput screen (the primary assay). We will use recombinant purified Kvbeta protein to screen for substrates in an enzymatic assay that follows the consumption of NADPH. Specific Aim 2. To test in an electrophysiological assay the positive hits from the screen (the secondary assay). We will test positive hits from Aim 1 using an electrophysiological assay that monitors channel functional change. Small-molecule substrates of Kvbeta will be used as a tool in our combined approaches of biochemistry, Xray crystallography, and electrophysiology to dissect molecular details of the coupling mechanism. The substrates will also provide a reasonable starting point for developing a new class K channel modulators.

描述（由申请人提供）：电压依赖性的钾通道（KV）是整体膜蛋白，在响应膜电压变化时，催化钾离子以扩散整个细胞膜。 KV通道调节膜兴奋性，对于许多生理过程，例如心脏的节奏跳动，神经元之间的交流和激素的分泌至关重要。在细胞中，KV通道始终与其他蛋白质组装在一起，通道功能通过这些相关蛋白质受细胞环境调节。潜在的Aldo-Keto还原酶（AKR）的β亚基（KVBETA）附着在KV1家族通道的细胞内侧。它们在大脑中无处不在，在心脏中充满表达，并与控制细胞兴奋性和感知细胞氧化还原态有关。这项研究的长期目标是了解KVBETA在细胞生物学中的作用。我们最近已经确定了两个KVBETA底物，并且首次证明KVBETA是功能性AKR。我们还发现，底物在灌注到通道的细胞内侧时，仅在共表达KVBETA时调节通道的功能。这些令人兴奋的新结果使我们假设KV1通道功能与KVBETA酶促活动耦合。在此提案中，我们着重于筛选KVBETA底物，并提出以下两个具体目的：特定目标1。通过高吞吐量屏幕（主要测定）识别KVBETA的小分子基板。我们将使用重组纯化的kVBETA蛋白来筛选底物，以鉴于NADPH消耗的酶法测定。特定目的2。在电生理测定中测试屏幕上的正命中（次要测定）。我们将使用监视通道功能变化的电生理测定法测试AIM 1的正命中。 KVBETA的小分子底物将用作我们的生物化学，X射线晶体学和电生理学的组合方法中的一种工具，以剖析耦合机制的分子细节。底物还将为开发新的K类调节器提供合理的起点。