Structural mechanism of K channel modulation by cellular redox state

细胞氧化还原态调节 K 通道的结构机制

• 批准号: 7081981
• 负责人: Ming Zhou
• 金额: $ 40.25万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-15 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7081981
• 关键词: NAD(H) phosphate; X ray crystallography; Xenopus; aldehyde /ketone oxidoreductase; conformation; crystallization; enzyme activity; intracellular; ion transport; membrane permeability; membrane potentials; oxidation reduction reaction; potassium channel; potassium ion; protein protein interaction; protein structure function; voltage gated channel; NAD(H) phosphate; X ray crystallography; Xenopus; aldehyde /ketone oxidoreductase; conformation; crystallization; enzyme activity; intracellular; ion transport; membrane permeability; membrane potentials; oxidation reduction reaction; potassium channel; potassium ion; protein protein interaction; protein structure function; 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. The beta subunit (Kv-beta) of the Shaker type Kv channels (Kv1) permanently attaches to the intracellular side of a channel and is implicated in channel modulation during oxidative stresses and hypoxic conditions. Sequence conservation suggests that Kv-beta resembles an aldo-keto reductase (AKR), and the crystal structure of a Kv-beta showed that it has a canonical AKR fold, a tightly bound cofactor nicotinamide adenine dinucleotide phosphate (NADPH), and highly conserved catalytic residues in the right geometry for catalysis to happen. However, the enzymatic activity of Kv-beta has never been demonstrated. The overall objectives of this proposal are to examine how Kv-beta as a functional AKR modulates channel function, to investigate how the enzymatic activity is coupled to channel activities, and to develop an atomic level understanding of the coupling mechanism. The long-term goals of the project are to understand the physiology of Kv-beta, and the principles governing Kv channel modulations. We have recently identified several Kv-beta substrates, and demonstrated that Kv-beta is a functional aldo-keto reductase. We also found that the substrates modulate channel function only when a Kv-beta is co- expressed. These exciting new results led us to hypothesize that: 1) the AKR function of Kv-beta is coupled to Kv channel functions; 2) the coupling is achieved through interactions between intracellular domains and Kv-beta 3) different redox states of Kv-beta have different conformations that induce a conformational change of a channel domain. To test these hypotheses, we propose the following three specific aims: Aim 1: To examine the functional coupling between channel activities and the AKR activity of Kv-beta. Aim 2: To investigate the molecular bases of the coupling mechanism. Aim 3: To investigate the structural bases of the coupling. Results from this project will help us understand modulations of the various Kv channel families, and will help develop therapeutic reagents that target the macromolecule complex.

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。 Shaker型KV通道（KV1）的β亚基（KV-BETA）永久连接到通道的细胞内侧，并与氧化应激和低氧条件下的通道调节有关。 Sequence conservation suggests that Kv-beta resembles an aldo-keto reductase (AKR), and the crystal structure of a Kv-beta showed that it has a canonical AKR fold, a tightly bound cofactor nicotinamide adenine dinucleotide phosphate (NADPH), and highly conserved catalytic residues in the right geometry for catalysis to happen.但是，从未证明KV-β的酶活性。该提案的总体目标是检查KV-beta作为功能AKR如何调节通道函数，以研究酶活性如何与信道活动耦合，并对耦合机制进行原子水平的理解。该项目的长期目标是了解KV-beta的生理学以及管理KV渠道调制的原则。我们最近已经确定了几个KV-β底物，并证明KV-beta是一种功能性的Aldo-Keto还原酶。我们还发现，仅在共同表达KV-beta时，底物调节通道函数。这些令人兴奋的新结果导致我们假设：1）KV-BETA的AKR函数与KV通道函数耦合； 2）耦合是通过细胞内结构域与KV-beta之间的相互作用实现的3）KV-β的不同氧化还原状态具有不同的构象，可诱导通道域的构象变化。为了检验这些假设，我们提出以下三个特定目的：目标1：检查通道活动与KV-BETA的AKR活性之间的功能耦合。目标2：研究耦合机制的分子碱基。目标3：研究耦合的结构底座。该项目的结果将有助于我们了解各种KV渠道家族的调制，并有助于开发针对大分子复合物的治疗试剂。