Pregnenolone constricts cerebral vascular arteries through the direct modulation of BK ion channels

孕烯醇酮通过直接调节 BK 离子通道收缩脑血管动脉

• 批准号: 10441131
• 负责人: Kelsey Cleland North
• 金额: $ 2.12万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-04-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10441131
• 关键词: 4-Aminopyridine; Address; Affinity; Alzheimer' s Disease; Anxiety; Area; Arteries; Arteriosclerosis; Binding Sites; Biological; Blood Vessels; Blood flow; Brain; Brain Diseases; Caliber; Cells; Cephalic; Cerebrum; Cholesterol; Cognition; Cognitive deficits; Coupling; Data; Differentiation and Growth; Diffuse; Docking; Doctor of Philosophy; Electrophysiology (science); Endothelium; Engineering; Female; Growth; Impaired cognition; In Vitro; Ion Channel; Ion Channel Protein; Knockout Mice; Knowledge; Lead; Ligands; Lipid Bilayers; Lipids; Mammals; Mediating; Membrane; Mental Depression; Methods; Modeling; Modification; Molecular; Molecular Target; Mus; Muscle Cells; Muscle Contraction; Mutation; Neuroglia; Neurons; Organ; Organelles; Organism; Peripheral; Pharmacology; Pharmacotherapy; Physiological; Physiology; Pregnenolone; Protein Subunits; Proteins; Regulation; Reporting; Research; Resolution; Rodent; Role; Side; Signal Transduction; Site; Smooth Muscle; Smooth Muscle Myocytes; Steroid Receptors; Steroids; Tail; Techniques; Testing; Tissues; Vascular Smooth Muscle; Voltage-Gated Potassium Channel; Work; base; cerebral artery; cerebrovascular; cerebrovascular pathology; constriction; dimer; in vivo; large-conductance calcium-activated potassium channels; male; middle cerebral artery; molecular modeling; mouse model; neurosteroids; patch clamp; paxilline; pressure; receptor; response; sex; vasoactive agent; voltage

In mammals, the Ca2+- and voltage-gated K+ channels of large conductance (BK) consist of a tetramer of channel-forming α and regulatory subunits. BK β1 subunits show localized expression in smooth muscle (SM) and increase the Ca2+-sensitivity of BK, allowing this channel to oppose depolarization-induced Ca2+ influx and limit SM contraction. Pregnenolone (PREG) is a local and circulating neurosteroid involved in modulating neuronal firing, growth and differentiation. Studies suggest that depression, anxiety, and Alzheimer progression could be modified through optimization of PREG levels. PREG effects on the brain have been primarily attributed to PREG actions on the neurons themselves. However, PREG is a vasoactive agent in peripheral arteries, exerting its effect via steroid receptors. Despite the vital role of optimal artery diameter for brain function and the fact that PREG is a vasoactive agent, studies of PREG on cerebral artery function are unavailable. My preliminary data support the idea that PREG reduces both SM BK function and middle cerebral artery (MCA) diameter independently of cytosolic/membrane receptors and downstream signaling. Rather, PREG actions seem to result from direct sensing of PREG by specific BK subunits and targeting of distinct gating mechanisms. Thus, I will study PREG actions on SM BK function and artery diameter using SM cells, de-endothelialized, electroporated MCA, engineered BKs introduced into cells and tissues, and will take advantage of BK subunit knockout mouse models. In a proposal that spans from molecular to organ resolution, I will address two Aims: A1) Specific BK subunits and gating mechanisms mediate PREG-induced inhibition of BK activity in MCA SM. Thus, I will establish whether: 1.1) PREG inhibition of vascular SM BK is enabled by PREG direct sensing by specific BK subunit(s) and a defined sensing site; 1.2) PREG sensitivity of BK is underlined by PREG disruption of distinct gating mechanisms; 1.3) the direct action of PREG on BK remains in intact SM cells. A2) PREG-induced MCA constriction results from steroid inhibition of SM BK. 2.1) Comparison of key findings in male vs. female, natural vs. electroporated MCA will address the roles of sex, BK subunits, and docking sites in PREG action. 2.2) I will confirm the ex-vivo data in-vivo using a cranial window. I will be the first to address the molecular targets and gating mechanisms mediating PREG action on brain vessels. Directed by Drs. Dopico and Bukiya at UTHSC, the research will provide key concepts and methods (single channel electrophysiology, Horrigan-Aldrich gating modeling, and rodent cranial window) to obtain my Ph.D. degree.

在哺乳动物中，具有大电导（BK）的Ca 2 +-和电压门控K+通道由一个 通道形成α和调节亚基的四聚体。BK β1亚基显示局部 在平滑肌（SM）中表达，并增加BK的Ca 2+敏感性，使该通道 对抗去极化诱导的Ca ~（2+）内流，限制SM收缩。孕烯醇酮（Preg） 一种局部和循环神经类固醇，参与调节神经元放电、生长和 分化研究表明，抑郁，焦虑和阿尔茨海默病的进展可能是 通过优化PREG水平进行修改。孕激素对大脑的影响主要是 归因于PREG对神经元本身的作用。然而，PREG是一种血管活性剂 在外周动脉中，通过类固醇受体发挥作用。尽管最佳的 动脉直径对脑功能的影响以及PREG是一种血管活性剂的事实， PREG对脑动脉功能的影响不可用。我的初步数据支持这样一种观点， PREG单独降低SM BK功能和大脑中动脉（MCA）直径 细胞溶质/膜受体和下游信号传导。相反，PREG的行动似乎 由特异性BK亚基直接感应PREG和靶向不同的门控产生 机制等因此，我将使用SM研究PREG对SM BK功能和动脉直径的作用 细胞，去内皮化的，电穿孔的MCA，引入细胞和组织的工程化BK， 并将利用BK亚基敲除小鼠模型。在一项提案中， 分子到器官的分辨率，我将解决两个目标：A1）特异性BK亚基和门控 机制介导PREG诱导的MCA SM中BK活性的抑制。因此，我将建立 是否：1.1）血管SM BK的PREG抑制通过特异性的PREG直接感测实现 BK亚基和确定的传感位点; 1.2）BK的PREG敏感性由PREG加下划线 不同门控机制的破坏; 1.3）PREG对BK的直接作用保持完整 SM细胞。A2）PREG诱导的MCA收缩由SM BK的类固醇抑制引起。2.1） 比较男性与女性、天然与电穿孔MCA的关键发现将解决 性别、BK亚基和对接位点在PREG作用中的作用。2.2)我会确认体外数据 在体内使用颅窗。我会第一个解决分子目标和门控问题 介导PREG对脑血管作用的机制。由Dopico博士和Bukiya博士执导， UTHSC，该研究将提供关键概念和方法（单通道电生理学， Horrigan-Aldrich门控建模和啮齿动物颅窗）获得博士学位。℃下