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

基本信息

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
项目类别：
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&apos 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

项目摘要

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，大电导（BK）的Ca2+和电压门控的K+通道由A组成通道形成α和调节亚基的四聚体。 BKβ1亚基显示局部在平滑肌（SM）中表达并增加BK的Ca2+ - 敏感性，允许此通道与去极化诱导的Ca2+影响并限制SM收缩。妊娠（PREG）是局部和循环的神经类固醇，参与调节神经元发射，生长和分化。研究表明，抑郁症，动画和阿尔茨海默氏症的发展可能是通过优化PREG水平进行修改。对大脑的preg影响是主要的归因于对神经元本身的预先作用。但是，PREG是血管活性剂在外周动脉中，通过类固醇受体发挥作用。尽管最佳作用至关重要大脑功能的动脉直径以及Preg是血管活性剂的事实，研究脑动脉功能的良性不可用。我的初步数据支持这样的想法 PREG独立降低SM BK功能和中大脑动脉（MCA）直径胞质/膜受体和下游信号传导。相反，预先行动似乎正在由特定的BK亚基和靶向不同门控的直接灵敏度产生机制。这是我使用SM研究对SM BK功能和动脉直径的预先作用细胞，电呈电动，电穿孔的MCA，引入细胞和组织的工程BK，并将利用BK亚基敲除鼠标模型。在跨越的提议中分子到器官分辨率，我将解决两个目标：A1）特定的BK亚基和门控机制介导了MCA SM中BK活性的预抑制。那我会建立是否：1.1）通过特定的preg直接敏感性启用了对血管SM BK的预抑制 BK亚基（S）和定义的灵敏度位点； 1.2）BK的PREG灵敏度由PREG强调破坏不同的门控机制； 1.3）PREG对BK的直接作用保持完整 SM细胞。 A2）由类固醇抑制SM BK引起的MCA预受限制。 2.1）在男性与女性，自然与电穿孔MCA中的关键发现的比较将解决性别，BK亚基和对接站点的作用。 2.2）我将确认前体数据在体内使用颅窗。我将是第一个解决分子靶标和门控的人介导对脑视频的预先作用的机制。由Drs执导。 Dopico和Bukiya在 UTHSC，该研究将提供关键概念和方法（单个通道电生理学， Horrigan-Aldrich门控建模和啮齿动物颅骨窗口）以获得我的博士学位。程度。