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

内源性类固醇(STS)的快速、非基因组效应，如调节局部动脉直径，已被证实 经典地归因于类固醇受体与下游信号的耦合。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能通过类固醇-离子通道蛋白直接相互作用来调节动脉内径，目前尚不清楚。离子通道的功能往往是通道形成和调节亚基协同作用的结果。平滑肌(SM)-丰富的β-1亚基与通道形成α结合，增加钙/电压门控钾通道(BK)的钙敏感性，使动脉SM-BK反馈去极化驱动的钙内流，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调节动脉内径不依赖ST受体，而是通过α和β1亚基上的特定位点直接感受ST。基于之前关于胆固醇和胆石酸盐的文献，以及初步的数据，我们将在基因工程小鼠的SM细胞中表达的重组通道上应用计算模型和膜片钳技术，以确定选定的ST与特定的BK氨基酸之间的分子相互作用，以及对与病理生理相关的血管：大脑中动脉、基底动脉和肠系膜动脉的直径进行评估，这些血管表达不同水平的β1。平行地，AIM 2(离子通道生物物理和药物化学)结合了药效团优化、化合物优先功能分析和机器人膜片钳、复杂门控分析、和计算对接，以获得两类不同的新型β1靶向非甾体类似物(NSTA)，它们呈现出与蛋白质相互作用的不同结构基础，从而激活含有β1的BK或反对这一作用。最后，从目标1和目标2获得的知识将被整合到目标3(翻译科学)中，在那里我们将证明在含有β1的BK上分别充当激动剂或拮抗剂的新型NSTA要么作为内皮非依赖性的动脉扩张剂，要么对抗这一作用，不同的动脉基于β1的表达表现出不同的敏感性。我们将通过展示选择性的新药物对抗或协同内源性STS的血管作用来进一步整合这些目标。最后，新药物的有效性将在动脉张力增加的疾病模型中进行测试，即自发性高血压大鼠。我们对大脑和外周动脉的关注与影响这两种血管的人类疾病的治疗最相关(例如，高血压脑病)。当内皮功能受损时，例如衰老或中风，独立于内皮发挥作用的基于β1的配体将特别有用。将计算方法与不同点突变的电生理学相结合作为一种策略，而不是耗时的结构方法，高通量膜片钳的使用，以及我们手中所有实验的可行性，确保了在R01奖的时间框架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.