Vasodilation via selective pharmacological targeting of BK channel beta1 subunits

描述（由申请人提供）：在动脉肌细胞中，Ca2+门控K+（BK）通道的激活限制了Ca2+内流，从而导致血管舒张。在大多数细胞中，BK通道由通道形成（1）和辅助（2）亚基组成。21亚型在血管肌细胞中高度表达，在其他细胞中几乎不存在，是限制血管肌细胞收缩的关键因素。因此，BK 21成为开发新型血管扩张剂的理想靶点。 几种类固醇激活BK通道，但其作用机制仍不清楚。类固醇的目标BK通道的变体亚基组合，这是否类固醇激活BK通过特定的对接通道上，而是次要的膜脂质的非特异性扰动的问题。我们最近发现胆烷类类固醇，如石胆酸盐（LC），选择性激活含21 BK，导致阻力大小的动脉扩张。值得注意的是，其他2个亚型（2 - 4）未能提供LC对BK通道的敏感性，这表明在21中存在胆烷类固醇识别区域。使用嵌合2s和计算机动力学，我们确定了两个候选网站在21类固醇识别。在本研究中，我们将利用计算机动力学、有机合成、点突变和膜片钳技术来确定胆烷甾体在BK 21上对接的实际位点和化学力。我们设计了选定的LC非甾体类似物（NSA），将用于定义对接位点的结构特征。在初步数据的支持下，将在突变的BK上探测比LC更有效地对接到该位点的NSA，以确定NSA作为通道激活剂的功效。一旦确定了配体对接位点和疗效（目标1），我们将结合联合收割机电压和电流钳方法，单通道动力学建模和共聚焦显微镜的研究，将从孤立膜的天然通道的综合方法，将评估BK电流，膜电位和局部Ca 2+信号在完整的心肌细胞之间的相互作用。这些研究将确定LC和NSA对接在BK 21上导致BK电流增加从而抑制肌细胞收缩性的作用机制（目的2）。 最后，使用加压、插管动脉、颅窗和评价体内肠系膜动脉直径变化，我们将确定含21 BK通道的激活对阻力大小、小动脉扩张的贡献，解决局部Ca2+和平滑肌膜电位在配体作用中的任何可能作用（目的3）。对于目标2和3，我们将利用BK 21 K/O小鼠模型。 该提案将带来关于类固醇位点和含21 BK通道的作用机制以及最终扩张阻力大小动脉的基本新信息。翻译潜力在于开发新的SA血管扩张剂，其通过选择性靶向BK 21起作用，独立于内皮，并且没有类固醇作用。由于高发病率的疾病需要急性扩张的肠系膜动脉和脑动脉，我们专注于第3 - 4支肠系膜动脉和阻力大小，大脑中动脉。 公共卫生相关性：细胞膜启动信号的重要性，生理类固醇越来越多地认识到，但类固醇作用于膜蛋白，包括离子通道的分子位点和机制，往往仍然未知。结合理论（单通道动力学建模，计算动力学）和实验在体外和体内啮齿动物模型，我们将确定BK通道21亚基的分子位点和胆甾类类固醇和非类固醇类似物（NSA）激活平滑肌BK通道的作用机制，从而扩张阻力大小的动脉。 结果将是至关重要的设计新的，类固醇副作用的NSA治疗流行的人类疾病，需要急性血管舒张的全身和脑动脉，独立于完整的内皮功能。

DESCRIPTION (provided by applicant): In arterial myocytes, activation of Ca2+-gated K+ (BK) channels limits Ca2+ influx and, thus, leads to vasodilation. In most cells, BK channels consist of channel-forming (1) and accessory (2) subunits. The 21 subtype is highly expressed in vascular myocytes and barely found in other cells, and serves as a key element to limit vascular myocyte contraction. Thus, BK 21 emerges as an ideal target to develop novel vasodilators. Several steroids activate BK channels, yet their mechanism of action remains unclear. Steroids target BK channels of variant subunit combinations, which questions whether steroids activate BK via specific docking on the channel or, rather, secondarily to nonspecific perturbation of membrane lipids. We recently found that cholane steroids, such as lithocholate (LC), selectively activate 21-containing BK, causing dilation of resistance -size arteries. Notably, other 2 subtypes (2-4) failed to provide LC sensitivity to BK channels, suggesting the existence of a cholane steroid-recognizing region in 21. Using chimeric 2s and computer dynamics, we identified two candidate sites in 21 for steroid recognition. In this proposal, we will use computer dynamics, organic synthesis, point mutagenesis and patch-clamp to identify the actual site and chemical forces involved in cholane steroid docking on BK 21. We designed selected LC nonsteroid analogs (NSA) that will be used to define the structural features of the docking site. Supported by preliminary data, NSA that dock onto this site more effectively than LC will be probed on mutated BK to determine NSA efficacy as channel activators. Once ligand docking site and efficacy are determined (Aim 1), we will combine voltage- and current-clamp methods, single channel kinetic modeling and confocal microscopy in studies that will go from native channels in isolated membranes to integrative approaches that will assess the interplay among BK current, membrane potential and local Ca2+ signals in intact myocytes. These studies will identify the mechanism of action by which LC and NSA docking on BK 21 leads to increased BK current and thus, depressed myocyte contractility (Aim 2). Finally, using pressurized, cannulated arteries, a cranial window, and evaluation of mesenteric artery diameter changes in vivo, we will determine the contribution of activation of 21-containing BK channels to dilation of resistance-size, small arteries, addressing any possible role of local Ca2+ and smooth muscle membrane potential in ligand action (Aim 3). For Aims 2 & 3 we will take advantage of the BK 21 K/O mouse model. The proposal will bring fundamental new information on steroid site and mechanisms of action on 21- containing BK channels and eventual dilation of resistance-size arteries. Translational potential resides on developing novel SA vasodilators that act via selective targeting of BK 21, independently of endothelium, and devoid of steroid actions. Given the high prevalence of disease requiring acute dilation of both mesenteric and cerebral arteries, we focus on 3rd-4th branches of mesenteric artery and resistance-size, middle cerebral artery. PUBLIC HEALTH RELEVANCE: The importance of cell membrane-initiated signaling by physiological steroids is increasingly recognized, yet the molecular site and mechanisms of steroid action on membrane proteins, including ion channels, often remain unknown. Combining theoretical (single channel kinetic modeling, computational dynamics) and experimental in vitro and in vivo rodent models, we will identify the molecular site in BK channel 21 subunit and mechanism of action by which cholane steroids and nonsteroid analogs (NSA) activate smooth muscle BK channels and, thus, dilate resistance-size arteries. Results will be critical to design novel, steroid side effect-free NSA to treat prevailing human diseases that require acute vasodilation of both systemic and cerebral arteries, independently of intact endothelial function.