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

在哺乳动物中，钙门控和电压门控的大电导钾通道(BK)由一个 通道形成α和调节亚基的四聚体。BKβ1亚单位显示本地化 在平滑肌(SM)中表达，并增加BK的钙敏感性，从而允许该通道 对抗去极化引起的钙内流，限制肌条收缩。孕烯醇酮(PREG)是 一种局部和循环中的神经类固醇，参与调节神经元的放电、生长和 差异化。研究表明，抑郁、焦虑和阿尔茨海默氏症的进展可能是 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是一种血管活性物质 在外周动脉中，通过类固醇受体发挥作用。尽管最佳方案的关键作用 大脑功能的动脉直径和PREG是血管活性物质的事实，研究 PREG对脑动脉功能的影响尚不清楚。我的初步数据支持这样的观点 PREG独立地降低SM BK功能和大脑中动脉(MCA)内径 胞浆/膜受体和下游信号转导。相反，PREG的行动似乎 由特定BK亚基直接感应PREG和靶向不同的门控所产生的结果 机械装置。因此，我将使用SM研究PREG对SM BK功能和动脉内径的作用 去内皮化的细胞，电穿孔的MCA，导入细胞和组织的工程化BKs， 并将利用BK亚单位基因敲除小鼠模型。在一项横跨 从分子到器官的解析，我将讨论两个目标：1)特定的BK亚基和门控 PREG抑制MCA SM中BK活性的机制因此，我将确立 1.1)PREG对血管SM BK的抑制作用是否通过特定的PREG直接感应来实现 BK亚基(S)和一个确定的感测位点；1.2)BK的PREG敏感性由PREG强调 破坏不同的门控机制；1.3)PREG对BK的直接作用仍然完好无损 SM细胞。A2)类固醇抑制SM-BK引起的大脑中动脉收缩。2.1) 男性与女性、自然MCA与电穿孔MCA的关键发现的比较将解决 性别、BK亚基和对接部位在PREG活动中的作用。2.2)我会确认体外数据 在活体内使用颅窗。我将是第一个解决分子目标和门控问题的人 PREG对脑血管作用的机制。由多比科博士和武基亚博士执导 UTHSC，这项研究将提供关键的概念和方法(单通道电生理， Horrigan-Aldrich门控建模和啮齿动物颅窗)以获得博士学位。

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.