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

在哺乳动物中，大电导Ca2+和电压门控K+通道（BK）由a

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.