ALLOSTERIC REGULATION OF ION CHANNEL GATING

描述(申请人提供)：这项工作的长期目标是了解自然产生的刺激和抑制物调节BK型钙(钙)激活钾(K+)通道的打开和关闭的潜在分子机制。BK通道与几乎所有其他离子通道的一个共同特征是，对蛋白质一个部分的生理刺激的感知与对该蛋白质另一部分发生的关键功能特性的调节是耦合的。在BK通道中，对膜电压或胞浆钙离子变化的感知调节着通过该通道的离子通量的激活。BK通道独立地对两个不同的生理信号作出反应，这对于研究连接的潜在分子步骤是一个优势 这些过程。了解这些过程很重要，原因有两个。首先，由于BK通道在许多生理系统中的重要作用，了解BK通道的调节有望为许多不同的疾病病理机制提供洞察力，并为疾病的改善提供策略。其次，通过利用BK通道的独特功能，这项工作将提供关于离子通道调节机制的新的一般性见解。这些洞察力有望有助于理解基本上所有离子通道和其他蛋白质的调节。本项目主要关注BK通道激活如何调控的两个主要方面。首先，将研究BK通道孔衬S6螺旋在定义通道浇口和耦合到浇口开口中的作用。BK S6螺旋已被证明在K+通道中是独一无二的，因为面对房水内孔的残基。作为推论，BK S6残基参与了独特的状态依赖的相互作用，有利于开放或封闭的构象。我们假设，定义这些相互作用将是理解BK通道机制的关键。利用电生理学和分子生物学相结合的方法，我们将探索BK S6残基与通道其他部分的相互作用，并研究S6残基的状态依赖性运动。第二，BK通道被一类致突变毒素所抑制。这些毒素是通道构象的有用探针，对这些毒素作用机制的研究有望为通道调节的新机制提供新的见解。这些毒素选择性地稳定关闭的通道状态。这类抑制机制的研究有望对其他一些离子通道的抑制具有广泛的意义。本工作对BK通道功能调控的理解具有潜在的医学意义，不仅因为BK通道是哮喘、癫痫、肿瘤生长和缺血性损伤的有希望的治疗靶点，而且还因为BK通道的病理变化可能是各种疾病状态的基础。

DESCRIPTION (provided by applicant): The long-term aim of this work is to understand the underlying molecular mechanisms by which naturally occurring stimuli and inhibitors regulate the opening and closing of the BK-type calcium (Ca2+)-activated potassium (K+) channel. A common characteristic shared by BK channels with essentially all other ion channels is that sensing of a physiological stimulus on one part of the protein is coupled to regulation of a key functional property occurring on another part of that protein. In the case of BK channels, sensing of either changes in membrane voltage or changes in cytosolic Ca2+ regulate the activation of ion flux through the channel. That BK channels respond independently to two distinct physiological signals is an advantage for investigation of the underlying molecular steps that link these processes. Understanding these processes is important for two reasons. First, because of the important role of BK channels in a number of physiological systems, understanding regulation of BK channels promises to provide insights into a number of different disease pathologies and provide strategies for diseases amelioration. Second, by taking advantage of unique features of the BK channel, the work will provide new general insights regarding the mechanisms of regulation of ion channels. Such insights promise to be of utility in understanding regulation of essentially all ion channels and other proteins. This project focuses on two major aspects of how activation of BK channels is regulated. First, the role of the BK channel pore-lining S6 helix in defining channel gating and coupling to gate opening will be examined. The BK S6 helix has been shown to be unique among K+ channels in terms of the residues that face the aqueous inner pore. As a corollary, BK S6 residues participate in unique state-dependent interactions favoring either open or closed conformations. We hypothesize that defining these interactions will be critical for understanding the BK channel machinery. Using methods of electrophysiology combined with molecular biology, we will probe the interactions of BK S6 residues with other parts of the channel and examine state- dependent movements of the S6 residues. Second, BK channels are inhibited by a family of tremorogenic toxins. These toxins are useful probes of channel conformation and investigation of the mechanism of action of these toxins promises to provide novel insight in a new mechanism of channel regulation. These toxins selectively stabilize closed channel states. Investigation of this category of inhibitory mechanism is expected to have broad significance for inhibition of a number of other ion channels. The understanding of regulation of BK channel function to be achieved in this work is of potential medical importance, not only because BK channels are promising therapeutic targets in asthma, epilepsy, tumor growth, and ischemic insults, but also because pathological alterations of BK channels may underlie various disease states.