Regulation of arterial diameter through specific sensing of endogenous steroids and novel nonsteroidal analogs by BK channel subunits

内源性类固醇（ST）的快速、非基因组效应，如局部动脉直径的调节，已被广泛研究。 经典地归因于与下游信号传导偶联的类固醇受体。在循环（激素）或局部（旁分泌神经类固醇）中达到nM-μM水平的内源性类固醇是否可以通过类固醇-离子通道蛋白直接相互作用调节动脉直径仍然是未知的。离子通道的功能通常是由通道形成亚基和调节亚基的协同作用引起的。平滑肌（SM）丰富的β1亚基与通道形成α相结合，增加Ca 2 +/电压门控K+通道（BK）的Ca 2+敏感性，使动脉SM BK反馈去极化驱动的Ca 2+内流，促进动脉扩张。目的1（脂质分子药理学和血管阻力生理学）验证了这一中心假设：内源性血管活性ST不依赖于ST受体调节动脉直径，而是通过α和β1亚基中的特定位点直接感受ST。基于先前关于胆固醇和石胆酸盐的出版物以及初步数据，我们将在来自基因工程小鼠的SM细胞中表达的重组通道上应用计算建模和膜片钳，以确定选定ST和特定BK氨基酸之间的分子相互作用，以及病理生理学相关性血管的直径评估：大脑中动脉、基底动脉和肠系膜动脉表达不同水平的β1。同时，目标2（离子通道生物物理学和药物化学）结合药效团优化，化合物优先级+功能测定与机器人膜片钳，复杂门控分析和计算对接，以获得两种不同类别的新型β1靶向非甾体类似物（NSTA），它们与蛋白质相互作用的结构基础不同，因此激活含β1的BK或对抗这种作用。最后，目标1和2获得的知识将被整合到目标3（翻译科学）中，我们将证明，作为含β1 BK的激动剂或拮抗剂的新型NSTA分别作为内皮非依赖性动脉扩张剂或对抗这种作用，不同的动脉显示出基于β1表达的不同敏感性。我们将进一步整合的目标，显示选择性，新的代理商，无论是抵消或协同血管的内源性ST的行动。最后，将在动脉张力增加的疾病模型中测试新药剂的功效，即，自发性高血压大鼠。我们对脑和外周动脉的关注与影响两种血管的人类疾病的治疗最相关（例如，高血压性脑病）。当内皮功能受损时，如衰老或中风，独立于内皮发挥作用的β1-基配体将特别有用。将计算方法与电生理学结合在不同的点突变体上作为一种策略，而不是耗时的结构方法，使用高通量膜片钳，以及我们手中所有实验的可行性，确保了在R 01奖项的时间范围内成功评估几个位点和配体。

Rapid, nongenomic effects of endogenous steroids (STs), such as regulation of local artery diameter, have been classically attributed to steroid receptors coupled to downstream signaling. Whether endogenous steroids that reach nM-μM levels in circulation (hormones) or locally (paracrine neurosteroids) can regulate artery diameter by steroid-ion channel protein direct interactions remains largely unknown. Ion channel function often results from concerted actions of channel-forming and regulatory subunits. Smooth muscle (SM)-abundant β1 subunits associate with channel-forming α to increase the Ca2+-sensitivity of Ca2+/voltage-gated K+ channels (BK), which allows arterial SM BK to feedback on depolarization-driven Ca2+ influx and promote artery dilation. Aim 1 (lipid Molecular Pharmacology and vascular resistance Physiology) tests this central hypothesis: endogenous vasoactive STs regulate arterial diameter independently of ST receptors but through direct sensing of ST by specific sites in α and β1 subunits. Based on previous publications with cholesterol and lithocholate, as well as preliminary data, we will apply computational modeling and patch-clamp on recombinant channels expressed in SM cells from genetically engineered mice, to identify the molecular interactions between selected STs and specific BK amino acids, plus diameter evaluation in vessels of pathophysiological relevance: middle cerebral, basilar and mesenteric arteries, which express different levels of β1. In parallel, Aim 2 (ion channel Biophysics and Medicinal Chemistry) combines pharmacophore optimization, compound prioritization+ functional assays with robotic patch-clamp, complex gating analysis, and computational docking to obtain two distinct classes of novel β1-targeting nonsteroidal analogs (NSTAs) that present different structural basis of interaction with the protein and thus either activate β1-containing BK or oppose this action. Finally, knowledge obtained with aims 1 and 2 will be integrated into Aim 3 (Translation Science), where we will demonstrate that the novel NSTAs that act as either agonists or antagonists on β1-containing BK respectively behave either as endothelium-independent, artery dilators or oppose this action, with different arteries displaying differential sensitivity based on β1 expression. We will further integrate the aims by showing that selective, new agents either counteract or synergize the vascular actions of endogenous STs. Finally, the efficacy of the new agents will be tested in a model of disease where the arterial tone is increased, i.e., the spontaneously hypertensive rat. Our focus on brain and peripheral arteries is most relevant to the therapy of human conditions that affect both vessels (e.g., hypertensive encephalopathy). β1-based ligands that act independently of the endothelium will be particularly useful when endothelial function is impaired, such as with ageing or stroke. The combination of computational methods with electrophysiology on different point mutants as a strategy opposed to time-consuming structural methods, the use of high-throughput patch-clamp, and the feasibility of all experiments in our hands ensure the successful evaluation of several sites and ligands in the timeframe of an R01 award.