Ethanol actions on slo channels from arteries vs. brain

急性暴露于脑动脉肌细胞和神经元上的BK(Slol)通道的活动被改变 酒精中毒期间血液中的乙醇(Etoh)水平，这种修饰导致了Etoh 生理学的微扰。乙醇在体内的作用可能需要药物介导的增强或抑制 BK频道。我们的长期目标是确定导致BK的分子机制和蛋白质位点 对乙醇的通道差异反应及其对大脑药物作用的贡献。这一知识 对于确定大脑脆弱性和设计治疗干预措施是必不可少的 通过BK通道靶向的酒精驱动的病理。尤其是乙醇对大脑动脉肌细胞的抑制作用 BK电流是酒精介导的脑动脉收缩的关键事件，这是一种酒精作用 不依赖循环和血管内皮细胞因子，可能与酒精所致脑血管病变有关 疾病和事故。肌细胞丰富的BK pi亚基(由KNCMB1编码)促进Slol(编码 通过乙醇对KCNMA1)通道的抑制和与2型兰诺定受体(RyR2)的偶联，这些蛋白质 构成控制大脑动脉张力的三联体，并直接成为Etoh的靶子。Aim1解决了 BK pI膜水平在脑动脉无水乙醇易损性中的作用 在不同程度上易受脑血管事件的影响。目的是在啮齿动物模型(大鼠，KCNMB1 K/0)上进行测试 小鼠)在体外使用两者(Western-blotting，蛋白质生物素化，ICC/IF显微镜，膜片钳，cDNA. 反向通透性和分离的心肌细胞和动脉)和活体方法(关闭颅窗， 活体显微镜)。由类似的模型和方法定义，并由计算补充 建模和药物化学，AIM2决定新药靶向BK pi是否拮抗 乙醇引起的脑动脉收缩。三种蛋白质中可能存在的乙醇敏感部位和区域 Bk pi(SubAim 3.1)、Slo1(SubAim 3.2)和RyR2(Aim 4)通过 计算模拟、诱变和体外电生理学，这些部位对生理学的影响 用相应的啮齿动物动脉(KCNMB1K/O、KCNMA1K/O小鼠)逆通透性建立模型。 相关性(请参阅说明)： 大脑动脉收缩通常是大脑状况和与以下疾病相关的事故的主要组成部分 酒精中毒。我们将确定在离子通道中介导乙醇识别的位置 乙醇诱导的脑动脉收缩，并利用这一信息指出脆弱性和 对抗酒精摄入引起的脑血管病理生理的治疗方法。

The activity of BK (slol) channels in brain artery myocytes and neurons is modified by acute exposure to ethanol (EtOH) levels reached in blood during alcohol intoxication, such modification contributing to EtOH perturbation of physiology. EtOH actions in the body may require drug-mediated potentiation or inhibition of BK channels. Our long-term goal is to identify molecular mechanisms and protein sites that lead to BK channel differential responses to EtOH and their contribution to drug actions on the brain. This knowledge is essential towards identification of brain vulnerability and design of therapeutic interventions in alcohol-driven pathology via BK channel-targeting. In particular, EtOH inhibition of cerebral artery myocyte BK current is the key event in alcohol-mediated cerebral artery constriction, an alcohol action that is independent of circulating and endothelial factors, and likely contributes to alcohol-induced cerebrovascular disease and accidents. The myocyte-abundant BK pi subunit (coded by KNCMB1) facilitates slol (coded by KCNMA1) channel inhibition by EtOH and coupling to type2 ryanodine receptors (RyR2), these proteins constituting a triad that controls cerebral artery tone and is directly targeted by EtOH. Aim1 addresses the role of BK p i membrane levels in EtOH vulnerability of brain arteries that irrigate distinct CNS regions and are differentially subject to cerebrovascular events. The aim is tested in rodent models (rat, KCNMB1 K/0 mouse) using both in vitro (Western-blotting, protein biotinylation, ICC/IF microscopy, patch-clamp, cDNA reverse-permeabilization, and isolated myocytes and arteries) and in vivo methods (closed cranial window, intravital microscopy). Defined by similar models and methods, and supplemented by computational modeling and medicinal chemistry, Aim2 determines whether BK pi-targeting by novel agents antagonizes cerebral artery constriction caused by EtOH. Putative EtOH-sensing sites and regions in the three proteins that constitute the EtOH target: BK pi (SubAim 3.1), slol (SubAim 3.2) and RyR2 (Aim 4) are identified via computational modeling, mutagenesis and in vitro electrophysiology, the impact of these sites on physiology being established by reverse-permeabilization of relevant rodent artery (KCNMB1 K/O, KCNMA1 K/O mice). RELEVANCE (See instructions): Cerebral artery constriction is often a main component of brain conditions and accidents associated with ethanol intoxication. We will identify the ethanol-recognition sites in the ion channels that mediate ethanol-induced cerebral artery constriction and use this information to point at vulnerability and therapeutics to counteract cerebrovascular pathophysiology caused by ethanol intake.