Testing a Novel Push-Pull Mechanism for Ca2+-Dependent Coupling in BK Channels

 描述(申请人提供)：高电导钙通道和电压激活K通道(Slo1或BK通道)分布广泛，具有多种生理功能。BK通道单独作为α亚基发挥作用，如在骨骼肌中，或与辅助β亚基(β1-β4)联合作用，在不同组织中赋予不同的功能特性。BK通道或其β亚单位缺陷或缺失与许多疾病过程有关，包括高血压、哮喘、自闭症、智力低下、肥胖和癫痫。了解BK通道激活的正常机制对于了解疾病中功能是如何改变的，并提供有助于开发可能的治疗方法的分子信息至关重要。Slo1的四个α亚基组装成一个具有膜内核心和细胞质尾巴的通道。该芯片由四个电压感应域(VSD)和一个孔栅域(PGD)组成。胞质尾巴形成一个大的细胞内门控环。CA2与门环的结合通过一种鲜为人知的从门环到核心的耦合机制激活PGD。BK通道上各种β亚基的作用部位和作用机制也鲜为人知。最近的两个进展将使我们能够应用新的方法来解决耦合和β亚单位作用的机制。第一个进展是在闭合和开放状态下获得门环的蛋白质晶体结构 这表明，Ca~(2+)与门环的结合导致VSD下的核心“推”，这是由于门环的四个α-B螺旋的升高，以及由于门环中杠杆臂的移动而同时对核心的PGD中的S6节段产生的“拉”。第二个进步是我们对隔离核心本身的分离和功能表达，它提供了一个工具来将观察到的功能分配给核心、门环或两者。基于这些进展和功能数据，我们假设一种新的推拉机制将依赖于钙的激活从门环耦合到核心。在目标1中，我们使用基于推拉假说的具有预期结果的突变，对钙依赖耦合的推拉假说进行批判性测试。在目标2中，我们使用这些改进来定位β1-β4亚单位在修改BK通道到核心、门控环或两者的门控时的作用部位。在目标3中，我们试图获得改变钙依赖偶联的突变门环的蛋白质晶体结构，以及β亚基的多肽与门环之间的接触部位的结构，以提供对机制的结构洞察。这些目标的完成将为理解门控环和核心之间的钙依赖耦合机制以及BK通道的β亚单位调控机制提供新的视角。推拉模型如果被发现与将要应用的关键测试一致，将需要对所提出的钙依赖耦合机制进行范式转换，从单一的主动耦合结构转变为双同时主动的推拉耦合结构。

 DESCRIPTION (provided by applicant): High conductance Ca2+ and voltage activated K+ channels (Slo1 or BK channels) are widely distributed and play numerous physiological roles. BK channels function as α subunits alone, as in skeletal muscle, or in association with auxiliary β subunits (β1- β4) where they confer diverse functional properties in different tissues. Defectve or missing BK channels or their β subunits have been associated with many disease processes including hypertension, asthma, autism, mental retardation, obesity, and epilepsy. Understanding the normal mechanism of activation of BK channels is crucial to understanding how function is altered in disease, and to provide molecular information that would be useful in developing possible therapies. The four α subunits of Slo1 assemble to form a channel with an intra-membrane Core and a cytoplasmic Tail. The Core consists of four voltage sensing domains (VSD) and a pore gate domain (PGD). The cytoplasmic Tails form a large intracellular gating ring. Ca2+ binding to the gating ring activates the PGD through a poorly understood coupling mechanism from gating ring to Core. Also poorly understood are the sites and mechanisms of action of the various β subunits on BK channels. Two recent advances will allow us to apply new approaches to resolve the mechanisms of coupling and β subunit action. The first advance is obtaining the protein crystal structures of the gating ring in the closed and open conformations, which suggests that Ca2+ binding to the gating ring induces a "push" to the Core under the VSDs resulting from elevation of the four alpha-B helices of the gating ring, and a simultaneous "pull" on the S6 segments in the PGD of the Core arising from movement of lever arms in the gating ring. The second advance was our isolation and functional expression of the isolated Core itself, which provides a tool to assign observed functions to Core, gating ring, or both. Based on these advances and functional data we hypothesize that a novel Push-Pull mechanism couples Ca2+-dependent activation from the gating ring to the Core. In Aim 1 we critically test the Push-Pull hypothesis for Ca2+-dependent coupling using mutations with expected outcomes based on the Push-Pull hypothesis. In Aim 2 we use these advances to localize the sites of action of β1- β4 subunits on modifying gating of BK channels to the Core, gating ring, or both. In Aim 3 we seek to obtain the protein crystal structures of the mutated gating rings that alter Ca2+-dependent coupling and also the structures of the sites of contact between peptides of β subunits and gating ring to provide structural insight into mechanism. The completion of these aims should provide new insight into the mechanism of Ca2+-dependent coupling between gating ring and Core, and also into the mechanisms for β subunit modulation of BK channels. The Push-Pull model, if found to be consistent with the critical tests to be applied, will necessitate a paradigm shift in the proposed mechanism of Ca2+-dependent coupling, from a single active coupling structure to dual simultaneously active Push-Pull coupling structures.