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 K+ 通道由通道形成 α 和调节亚基 BK β1 亚基的四聚体显示出局部化。平滑肌 (SM) 中的表达并增加 BK 的 Ca2+ 敏感性，从而允许该通道对抗去极化引起的 Ca2+ 流入并限制 SM 收缩。一种局部循环神经类固醇，参与调节神经元放电、生长和研究表明抑郁症、焦虑症和阿尔茨海默病的进展可能与此有关。主要通过优化 PREG 水平对大脑的影响进行修改。归因于 PREG 对神经元本身的作用然而，PREG 是一种血管活性剂。尽管最佳作用至关重要，但在外周动脉中，通过类固醇受体发挥其作用。动脉直径对大脑功能的影响以及 PREG 是一种血管活性剂的事实，我的初步数据支持这样的观点：关于脑动脉功能的 PREG。 PREG 独立降低 SM BK 功能和大脑中动脉 (MCA) 直径相反，PREG 的作用似乎是胞质/膜受体和下游信号传导的。由特定 BK 亚基直接感测 PREG 并针对不同门控而产生因此，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、电穿孔的 MCA、引入细胞和组织的工程 BK、并将在一项提案中利用 BK 亚基敲除小鼠模型。分子到器官的分辨率，我将解决两个目标：A1）特定的 BK 亚基和门控介导 PREG 诱导的 MCA SM 中 BK 活性抑制的机制因此，我将建立。是否： 1.1) PREG 对血管 SM BK 的抑制是通过特定的 PREG 直接传感实现的 BK 亚基和确定的传感位点；1.2) PREG 强调了 BK 的 PREG 敏感性； 1.3) PREG 对 BK 的直接作用保持不变； SM 细胞。PREG 诱导的 MCA 收缩是 SM BK 2.1) 类固醇抑制的结果。比较男性与女性、自然与电穿孔 MCA 的主要发现将解决以下问题性别、BK 亚基和对接位点在 PREG 作用中的作用 2.2) 我将确认离体数据。我将是第一个使用颅窗进行体内研究的人。介导 PREG 对脑血管作用的机制，由 Dopico 和 Bukiya 博士指导。 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）以获得博士学位。