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

 描述（由申请人提供）：高电导Ca 2+和电压激活的K+通道（Slo 1或BK通道）广泛分布，并发挥多种生理作用。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）联合发挥作用，在不同组织中赋予不同的功能特性。BK通道或其β亚基的缺陷或缺失与许多疾病过程相关，包括高血压、哮喘、自闭症、精神发育迟滞、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如何改变以及提供有助于开发可能疗法的分子信息至关重要。Slo 1的四个α亚基组装形成具有膜内核心和细胞质尾的通道。核心由四个电压传感域（VSD）和一个孔门域（PGD）组成。细胞质尾形成一个大的细胞内门控环。Ca 2+结合到门控环通过从门控环到核心的尚不清楚的偶联机制激活PGD。对BK通道上各种β亚基的作用位点和机制也知之甚少。最近的两项进展将使我们能够应用新的方法来解决耦合和β亚基作用的机制。第一个进展是获得了门控环在封闭和开放状态下的蛋白质晶体结构 这表明Ca 2+与门控环的结合诱导了由门控环的四个α-B螺旋的升高引起的VSD下的核心的“推”，以及由门控环中的杠杆臂的运动引起的核心的PGD中的S6区段上的同时的“拉”。第二个进步是我们对分离的核心本身的分离和功能表达，这提供了一种工具，可以将观察到的功能分配给核心、门控环或两者。基于这些进展和功能数据，我们假设一种新的推拉机制耦合钙依赖性激活从门控环的核心。在目标1中，我们严格测试推拉假说的钙依赖性耦合使用突变与预期结果的基础上推拉假说。在目标2中，我们使用这些进展定位β1- β4亚基在将BK通道的门控修饰到核心、门控环或两者上的作用位点。在目标3中，我们试图获得改变Ca 2+依赖性偶联的突变门控环的蛋白质晶体结构，以及β亚基肽与门控环之间的接触位点的结构，以提供对机制的结构洞察。这些目标的实现将为门控环和Core之间的Ca ~（2+）依赖性偶联机制以及BK通道β亚基调节机制提供新的见解。推拉模型，如果发现是一致的关键测试应用，将需要一个范式转变的Ca 2+依赖性耦合的拟议机制，从一个单一的主动耦合结构的双重同时积极推拉耦合结构。

 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.