Structural mechanism of K channel modulation by cellular redox state

描述（由申请人提供）：电压依赖性钾通道（Kv）是一种整合的膜蛋白，响应膜电压变化，催化钾离子扩散穿过细胞膜。KV通道调节细胞膜的兴奋性，在心脏节律性跳动、神经元间的通讯、激素分泌等生理过程中起着重要作用。Shaker型Kv通道（Kv 1）的β亚基（Kv-beta）永久附着于通道的细胞内侧，并在氧化应激和缺氧条件下参与通道调节。序列保守性表明Kv-β类似于醛酮还原酶（AKR），Kv-β的晶体结构表明它具有典型的AKR折叠，紧密结合的辅因子烟酰胺腺嘌呤二核苷酸磷酸（NADPH）和高度保守的催化残基，其几何形状正确，催化作用发生。然而，Kv-β的酶活性从未得到证实。该提案的总体目标是研究Kv-β作为功能性AKR如何调节通道功能，研究酶活性如何与通道活性偶联，并对偶联机制进行原子水平的理解。该项目的长期目标是了解Kv-β的生理学，以及Kv通道调制的原理。我们最近已经确定了几个Kv-β底物，并证明Kv-β是一种功能性醛酮还原酶。我们还发现，只有当Kv-β共表达时，底物才能调节通道功能。这些令人兴奋的新结果使我们假设：1）Kv-β的AKR功能与Kv通道功能偶联; 2）偶联是通过细胞内结构域和Kv-β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，诱导通道结构域的构象变化。为了验证这些假设，我们提出了以下三个具体目标：目标1：检查通道活动和Kv-β的AKR活动之间的功能耦合。目的2：探讨偶联机理的分子基础.目的3：研究耦合的结构基础。该项目的结果将有助于我们了解各种Kv通道家族的调节，并将有助于开发针对大分子复合物的治疗试剂。

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.