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

• 批准号: 7992134
• 负责人: ALEX M. DOPICO
• 金额: $ 39.01万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7992134
• 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; Clinical; Complex; Computer Simulation; Computers; Confocal Microscopy; Coupling; Data; Depressed mood; Development; Disease; Docking; Drug Industry; Elements; Endothelium; Environment; Evaluation; Figs - dietary; Future; Genbank; Genomics; High Prevalence; Image; In Vitro; Ion Channel; Ions; Kinetics; Lead; Ligands; Lipids; Mediating; Membrane; Membrane Lipids; Membrane Potentials; Membrane Proteins; Mesenteric Arteries; Mesentery; 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

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.

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通道由通道形成亚基(1)和辅助亚基(2)组成。21亚型在血管肌细胞中高表达，在其他细胞中很少发现，并且是限制血管肌细胞收缩的关键因素。因此，bk21成为开发新型血管扩张剂的理想靶点。几种类固醇可激活BK通道，但其作用机制尚不清楚。类固醇靶向不同亚基组合的BK通道，这就质疑类固醇是通过通道上的特异性对接激活BK，还是继发于膜脂的非特异性扰动。我们最近发现胆甾类类固醇，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个亚型（2-4）未能对BK通道提供LC敏感性，这表明21中存在胆烷类固醇识别区域。利用嵌合2s和计算机动力学，我们在21中确定了两个候选的类固醇识别位点。在本课题中，我们将利用计算机动力学、有机合成、点诱变和膜片钳来确定胆烷类固醇对接bk21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），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更有效地停靠到该位点的NSA，以确定NSA作为通道激活剂的功效。一旦确定配体对接位置和有效性（目的1），我们将结合电压和电流钳法，单通道动力学建模和共聚焦显微镜的研究，将从孤立膜中的天然通道到综合方法，评估完整肌细胞中BK电流，膜电位和局部Ca2+信号之间的相互作用。这些研究将确定LC和NSA对接bk21导致BK电流增加从而抑制肌细胞收缩力的作用机制（目的2）。最后，使用加压、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21-含BK通道的激活对阻力大小的小动脉扩张的贡献，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和目标3，我们将利用bk21 K/O小鼠模型。该建议将带来类固醇部位和作用机制的基本新信息，包括21- BK通道和阻力大小动脉的最终扩张。翻译潜力在于开发新的SA血管扩张剂，通过选择性靶向bk21起作用，独立于内皮细胞，不含类固醇作用。鉴于需要急性扩张肠系膜动脉和大脑动脉的疾病的高患病率，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。