Vasodilation via selective pharmacological targeting of BK channel beta1 subunits

描述(申请人提供)：在动脉肌细胞中，钙门控K+(BK)通道的激活限制了钙内流，从而导致血管扩张。在大多数细胞中，BK通道由通道形成(1)和附属(2)亚基组成。21亚型在血管肌细胞中高表达，在其他细胞中很少表达，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，但它们的作用机制尚不清楚。类固醇以不同亚基组合的BK通道为靶点，这就质疑类固醇是否通过特定的通道对接激活BK，或者更确切地说，是通过非特异性的膜脂扰动来激活BK。我们最近发现胆烷类固醇，如石胆酸盐(LC)，选择性地激活含有21-BK的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)不能提供对BK通道的LC敏感性，这表明在21个亚型中存在胆烷类固醇识别区域。利用嵌合体2S和计算机动力学，我们确定了21个候选位点中的两个类固醇识别。在这项提案中，我们将使用计算机动力学、有机合成、点突变和膜片钳来确定胆烷类固醇在BK 21上对接的实际位置和化学力。我们设计了精选的LC非类固醇类似物(NSA)，用于定义对接部位的结构特征。在初步数据的支持下，比LC更有效地对接到这个位置的NSA将被探测到突变的BK上，以确定NSA作为通道激活剂的有效性。一旦确定了配体对接的位置和有效性(目标1)，我们将结合电压和电流钳方法、单通道动力学建模和共聚焦显微镜进行研究，这些研究将从分离的膜上的天然通道转向综合方法，评估BK电流、膜电位和完整心肌细胞局部钙信号之间的相互作用。这些研究将确定LC和NSA对接BK 21导致BK电流增加从而抑制心肌细胞收缩的作用机制(目标2)。最后，使用加压的中空动脉、颅窗和评估活体肠系膜动脉内径的变化，我们将确定含有21-BK通道的激活对阻力大小的小动脉扩张的贡献，解决局部钙离子和平滑肌膜电位在配基作用中的任何可能作用(目标3)。对于AIMS 2和AIMS 3，我们将利用BK 21 K/O小鼠模型。该提案将带来有关类固醇在21-BK通道上的作用部位和作用机制以及最终阻力大小动脉扩张的基本新信息。翻译的潜力在于开发新的SA血管扩张剂，这种扩张剂通过选择性靶向BK 21发挥作用，不依赖于内皮，并且没有类固醇作用。鉴于肠系膜动脉和脑动脉急性扩张的发病率很高，我们将重点放在肠系膜动脉的第3-4支和阻力大小的大脑中动脉。

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.