Ethanol actions on slo channels from arteries vs. brain

脑动脉肌细胞和神经元中BK（soll）通道的活性通过急性暴露于 酒精中毒期间血液中达到的乙醇（EtOH）水平，这种改变有助于EtOH 生理学的扰动。EtOH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 通道对EtOH的差异反应及其对药物作用于大脑的贡献。这些知识 对于识别大脑脆弱性和设计治疗干预至关重要， 酒精驱动的病理通过BK通道靶向。特别是，EtOH抑制脑动脉肌细胞 BK电流是酒精介导的脑动脉收缩的关键事件，酒精的作用是 不依赖于循环和内皮因子，并可能导致酒精诱导的脑血管疾病 疾病和事故。肌细胞丰富的BK pi亚基（由KNCMB 1编码）促进sol（编码 通过KCNMA 1）通道抑制EtOH和耦合到2型兰尼碱受体（RyR 2），这些蛋白质 构成控制脑动脉张力的三联体，并被EtOH直接靶向。Aim 1解决了 BK β i膜水平在灌注不同CNS区域的脑动脉EtOH易损性中的作用， 脑血管事件的发生率不同在啮齿动物模型（大鼠，KCNMB 1 K/0）中测试该目的 小鼠），使用两种体外方法（蛋白质印迹、蛋白质生物素化、ICC/IF显微术、膜片钳、cDNA 反向透化，和分离的肌细胞和动脉）和体内方法（闭合颅窗， 活体显微术）。由类似的模型和方法定义，并辅以计算 Aim 2确定新药物的BK pi靶向是否拮抗 乙醇引起的脑动脉收缩。三种蛋白质中的推定EtOH敏感位点和区域 构成EtOH靶点：BK pi（SubAim 3.1）、sol 1（SubAim 3.2）和RyR 2（Aim 4）通过以下方法鉴定： 计算建模，诱变和体外电生理学，这些网站对生理学的影响 通过对相关啮齿动物动脉（KCNMB 1 K/O、KCNMA 1 K/O小鼠）进行反向透化来建立。 相关性（参见说明）： 脑动脉收缩通常是脑疾病的主要组成部分， 酒精中毒我们将确定离子通道中的乙醇识别位点， 乙醇诱导的脑动脉收缩，并使用此信息来指出脆弱性， 治疗剂，以抵消由乙醇摄入引起的脑血管病理生理学。

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.