ALLOSTERIC REGULATION OF ION CHANNEL GATING

描述（由申请人提供）：这项工作的长期目标是了解天然存在的刺激物和抑制剂调节BK型钙（Ca2+）激活钾（K+）通道开放和关闭的潜在分子机制。BK通道与基本上所有其他离子通道共有的共同特征是，在蛋白质的一部分上的生理刺激的感测与在该蛋白质的另一部分上发生的关键功能特性的调节相耦合。在BK通道的情况下，感测膜电压的变化或胞质Ca 2+的变化调节通过通道的离子通量的激活。BK通道对两种不同的生理信号的独立反应对于研究将两种不同的生理信号联系起来的潜在分子步骤是有利的。 这些过程。了解这些过程很重要，原因有二。首先，由于BK通道在许多生理系统中的重要作用，理解BK通道的调节有望提供对许多不同疾病病理学的见解，并提供改善疾病的策略。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见解。这些见解有望在理解基本上所有离子通道和其他蛋白质的调控方面发挥作用。该项目的重点是如何激活BK通道的调节的两个主要方面。首先，BK通道孔内衬S6螺旋在定义通道门控和耦合到门开的作用将被检查。BK S6螺旋已被证明是唯一的K+通道中的残基，面对水的内孔。作为一个推论，BK S6残基参与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至关重要的了解BK通道机制。利用电生理学与分子生物学相结合的方法，我们将探测BK S6残基与通道其他部分的相互作用，并检查S6残基的状态依赖性运动。第二，BK通道被致震颤毒素家族抑制。这些毒素是通道构象的有用探针，并且这些毒素的作用机制的调查有望在通道调节的新机制中提供新的见解。这些毒素选择性地稳定封闭通道状态。这类抑制机制的研究预期对抑制许多其他离子通道具有广泛的意义。在这项工作中要实现的BK通道功能的调节的理解是潜在的医学重要性，不仅因为BK通道在哮喘，癫痫，肿瘤生长和缺血性损伤的有前途的治疗靶点，而且因为BK通道的病理改变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.