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)是一种完整的膜蛋白，它响应于膜电压的变化，催化钾离子在细胞膜上扩散。KV通道调节细胞膜的兴奋性，在许多生理过程中都是必不可少的，如心脏的节律性跳动、神经元之间的通讯和激素的分泌。Shaker型Kv通道(KV1)的β亚基(Kv-beta)永久地附着在通道的胞内侧，参与氧化应激和低氧条件下的通道调节。序列保守表明Kv-β类似于醛酮还原酶(AKR)，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)，以及高度保守的催化残基，适合催化发生。然而，Kv-β的酶活性从未被证实。这项建议的总体目标是研究Kv-β作为一种功能性AKR是如何调节通道功能的，研究酶活性如何与通道活性耦合，并发展对耦合机制的原子水平的理解。该项目的长期目标是了解Kv-beta的生理学，以及控制Kv通道调制的原理。我们最近已经鉴定了几种Kv-beta底物，并证明Kv-beta是一种功能性的醛酮还原酶。我们还发现，只有当Kv-β共表达时，底物才能调制通道功能。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)这种偶联是通过细胞内结构域与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，从而导致通道结构域的构象变化。为了验证这些假说，我们提出了以下三个具体目标：目标1：检验通道活动和Kv-β的AKR活性之间的功能耦合。目的2：探讨偶联机制的分子基础。目的3：探讨联轴器的结构基础。这个项目的结果将帮助我们了解各种Kv通道家族的调节，并将有助于开发针对大分子复合体的治疗试剂。

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.