Structural mechanism of K channel modulation by cellular redox state

电压依赖性钾通道(Kv)是一种完整的膜蛋白，对膜的反应 电压的变化，催化钾离子在细胞膜上扩散。千伏通道调节 膜的兴奋性，在许多生理过程中都是必不可少的，例如 心脏、神经元之间的交流和荷尔蒙的分泌。β亚基(KVP) 振荡器类型Kv通道(KV1)永久附着在通道的胞内侧，并被牵连 在氧化应激和低氧条件下的通道调节。序列守恒表明 KVP类似于醛酮还原酶(AKR)，KVP的晶体结构表明它具有 典型的AKR折叠，一个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在正确的几何构型中，有利于催化发生。然而，酶 KVP的活性从未被证明过。这项提案的总体目标是研究KVP如何 作为一个功能性的AKR调节通道功能，为了研究酶活性是如何偶联到 通道活动，并发展原子水平的耦合机制的理解。长期的 该项目的目标是了解KVP的生理学，以及管理KV通道的原则 调制。我们最近鉴定了几种KVP底物，并证明了KVP是一种功能性的 醛酮还原酶。我们还发现，只有当KVP和KVP共同作用时，底物才能调制通道。 表达。这些令人兴奋的新结果使我们假设：1)KVP的AKR功能与 KV通道功能；2)细胞内结构域与KVP相互作用实现偶联 3)KVP的不同氧化还原状态具有不同的构象，从而导致a的构象变化 通道域。为了验证这些假设，我们提出了以下三个具体目标： 目的1：研究通道活动与KVP的AKR活动之间的功能偶联关系。 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通道家族的调制，并将有助于 开发针对大分子复合体的治疗试剂。

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 (Kvp) 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 Kvp resembles an aldo-keto reductase (AKR), and the crystal structure of a Kvp 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 Kvp has never been demonstrated. The overall objectives of this proposal are to examine how Kvp 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 Kvp, and the principles governing Kv channel modulations. We have recently identified several Kvp substrates, and demonstrated that Kvp is a functional aldo-keto reductase. We also found that the substrates modulates channel function only when a Kvp is co- expressed. These exciting new results led us to hypothesize that: 1) the AKR function of Kvp is coupled to Kv channel functions; 2) the coupling is achieved through interactions between intracellular domains and Kvp 3) different redox states of Kvp 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 Kvp. 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.