Vasodilation via selective pharmacological targeting of BK channel beta1 subunits

通过选择性药理学靶向 BK 通道 β1 亚基实现血管舒张

基本信息

批准号：
8600967
负责人：
ALEX M. DOPICO
金额：
$ 38.11万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2015-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8600967
关键词：
Acute Address Adverse effects Animal Model Area Arteries Back Basic Science Biology Blood Vessels C57BL/6 Mouse Caliber Cell membrane Cells Cephalic Cerebrum Chemicals Chinese Traditional Medicine Cholanes Clinical Complex Computer Simulation Computers Confocal Microscopy Coupling Data Depressed mood Development Disease Docking Drug Industry Elements Endothelium Environment Evaluation Future Genbank Genomics High Prevalence Ideal 1 Image In Vitro Ion Channel Ions Kinetics Lead Ligands Lipids Mediating Membrane Membrane Lipids Membrane Potentials Membrane Proteins Mesenteric Arteries Metabolism Methods Modeling Modification Molecular Mus Muscle Cells Muscle Fibers Mutagenesis Mutate Nuclear Organic Chemistry Organic Synthesis Peripheral Peripheral Resistance Pharmacology Pharmacotherapy Phenotype Physiological Process Proteins Proteolipids Rattus Recombinants Research Resistance Rest Rodent Rodent Model Role Secondary to Signal Transduction Site Smooth Muscle Steroid Receptors Steroids Structure-Activity Relationship System Testing Tissues Variant Vascular Smooth Muscle Vascular resistance Vasodilation Vasodilator Agents analog base cerebral artery design feeding human disease in vivo large-conductance calcium-activated potassium channels meetings middle cerebral artery molecular site mouse model novel patch clamp pharmacophore public health relevance research study stem structural biology therapeutic target trafficking transcription factor voltage

项目摘要

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.

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 Ca2+ 流入，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表达，在其他细胞中几乎没有发现，是限制血管肌细胞收缩的关键元件。因此，BK 21 成为开发新型血管扩张剂的理想靶标。多种类固醇可激活 BK 通道，但其作用机制仍不清楚。类固醇靶向不同亚基组合的 BK 通道，这质疑类固醇是否通过特定对接在通道上激活 BK，或者更确切地说，其次是膜脂的非特异性扰动。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 的 BK，导致阻力大小的动脉扩张。值得注意的是，其他 2 种亚型 (2-4) 未能提供 LC 对 BK 通道的敏感性，表明 21 中存在胆烷类固醇识别区域。利用嵌合 2 和计算机动力学，我们在 21 中确定了两个用于类固醇识别的候选位点。在本提案中，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 BK 21 上胆烷类固醇对接所涉及的实际位点和化学力。我们设计了选定的 LC 非类固醇类似物 (NSA)，它将用于定义对接站点的结构特征。在初步数据的支持下，比 LC 更有效地对接到该位点的 NSA 将在突变的 BK 上进行探测，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功效（目标 1），我们将在研究中结合电压和电流钳方法、单通道动力学建模和共焦显微镜，从分离膜中的天然通道到评估相互作用的综合方法完整肌细胞中的 BK 电流、膜电位和局部 Ca2+ 信号之间的关系。这些研究将确定 LC 和 NSA 对接 BK 21 导致 BK 电流增加，从而降低心肌细胞收缩力的作用机制（目标 2）。最后，使用加压的插管动脉、颅窗和体内肠系膜动脉直径变化的评估，我们将确定含有 21 的 BK 通道的激活对阻力大小的小动脉扩张的贡献，解决任何可能的作用配体作用中的局部 Ca2+ 和平滑肌膜电位（目标 3）。对于目标 2 和 3，我们将利用 BK 21 K/O 鼠标模型。该提案将带来关于类固醇位点和 21 含有 BK 通道的作用机制以及阻力大小动脉的最终扩张的基本新信息。转化潜力在于开发新型 SA 血管舒张剂，其通过选择性靶向 BK 21 发挥作用，独立于内皮细胞，并且没有类固醇作用。鉴于需要肠系膜动脉和脑动脉急性扩张的疾病的高患病率，我们重点关注肠系膜动脉的第 3-4 分支和阻力大小的大脑中动脉。