Ethanol actions on slo channels from arteries vs. brain

描述（由申请人提供）：急性乙醇暴露对电压门控和Ca 2+门控钾通道（BK）的调节涉及已知在酒精中毒期间改变的几种生理过程。在某些情况下，乙醇在体内的作用需要药物介导的BK激活，而在其他情况下，乙醇抑制BK来改变组织功能。我们研究的长期目标是确定决定BK的不同乙醇反应的分子机制和靶点，以及这种调节对体内急性乙醇作用的贡献。这一目标将有助于解决一个长期存在的谜团，即乙醇对离子通道的作用机制，并将导致酒精中毒的合理治疗干预。我们最近发现，乙醇对BK的作用是由通道形成亚基（Ca 2+）、BK天然配体（Ca 2+）和药物之间的基本相互作用引起的，但其他几个因素，如Ca 2+、BK辅助亚基（2）和通道复合物周围的脂质环境的翻译后修饰，能够微调最终的乙醇效应。我们还发现，酒精在酗酒期间在循环中获得的浓度，已知会增加中风的风险，通过减少脑动脉肌细胞BK电流引起脑血管收缩。然而，乙醇对脑血管作用的机制和分子靶点仍不清楚。脑动脉肌细胞BK是由B1和B2紧密结合的结果，B1控制BK的Ca 2+敏感性和与Ryanodine受体（RyR）的偶联。RyR产生火花，激活BK的局部Ca 2+信号。该提议的中心假设是，B1，通过控制BK-RyR耦合和BK-RyR的Ca 2+敏感性，是导致乙醇抑制BK电流的关键因素，因此，脑动脉收缩。我们将测试3个具体目标（A）。在A1中，我们将使用大鼠和小鼠模型（包括B1 K/O小鼠），测定乙醇对脑动脉张力的作用，体外电生理学和药理学，以测试乙醇诱导的动脉收缩和天然细胞中BK电流的减少是否需要B1。在A2中，我们将使用突变的Bs，单通道记录和动力学建模来确定B1使乙醇直接抑制BK的亚基结构域和机制。在A3中，我们将使用膜片钳和脂质双层电生理学，选择性抗体，共聚焦Ca 2+成像和药理学来确定乙醇是否抑制RyR，从而降低BK功能。在项目期结束时，我们预计已经确定了导致乙醇诱导的脑血管收缩的分子靶点和机制，这是与酒精中毒有关的脑血管疾病的基本要素。公共卫生相关性：在美国，酗酒是酒精滥用的主要形式。酗酒可能导致脑动脉收缩、血管痉挛和中风，所有这些都是乙醇引起的脑动脉平滑肌收缩的后果。我们发现，这种乙醇的行动是介导的平滑肌离子通道的BK型，这是复杂的异源寡聚蛋白。本研究将明确乙醇抑制BK电流和脑动脉收缩的蛋白亚基位点和分子机制。该提案将为设计合理的药物干预措施以治疗与酒精中毒相关的脑血管疾病提供重要信息。

DESCRIPTION (provided by applicant): Modulation of voltage- and Ca2+-gated potassium channels (BK) by acute ethanol exposure is involved in several physiological processes known to be altered during alcohol intoxication. In some cases, ethanol action in the body requires drug-mediated BK activation, while in others ethanol inhibits BK to modify tissue function. The long- term goal of our research is to pinpoint the molecular mechanisms and targets that determine differential ethanol responses of BK and the contribution of such modulation to acute ethanol actions in the body. This goal will help to address a long-standing enigma, that is, the mechanism of ethanol action on ion channels, and will lead to rational therapeutic interventions in alcohol intoxication. We recently showed that ethanol actions on BK result from a basic interaction among the channel-forming (slo) subunit, the BK natural ligand (Ca2+) and the drug, yet several other elements, such as posttranslational modification of slo, BK accessory subunits (2) and the lipid environment around the channel complex are able to fine-tune the final ethanol effect. We also showed that ethanol at concentrations obtained in circulation during binge drinking and known to increase the risk for stroke, causes cerebrovascular constriction by reducing cerebral artery myocyte BK currents. However, the mechanisms and molecular targets of ethanol action on cerebral vessels remain unknown. Cerebral artery myocyte BK result from the tight association of slo and B1, the latter controlling BK Ca2+ sensitivity and coupling to ryanodine receptors (RyR). RyR generates sparks, a local Ca2+ signal that activates BK. The central hypothesis of this proposal is that B1, by controlling slo Ca2+ sensitivity and BK-RyR coupling, is the key element that leads to ethanol inhibition of BK current and, thus, cerebral artery constriction. We will test 3 specific aims (A). In A1, we will use rat and mouse models (including B1 K/O mice), determination of ethanol action on cerebral artery tone, in vitro electrophysiology, and pharmacology to test whether ethanol-induced arterial constriction and reduction of BK current in native cells require B1. In A2, we will use mutated Bs, single channel recordings and kinetic modeling to pinpoint subunit domain and mechanism by which B1 enables ethanol direct inhibition of BK. In A3, we will use patch-clamp and lipid bilayer electrophysiology, selective antibodies, confocal Ca2+ imaging and pharmacology to determine whether ethanol inhibits RyR and, thus, decreased BK function. At the end of the project period, we expect to have identified both molecular target and mechanism leading to ethanol- induced cerebrovascular constriction, the fundamental element in cerebrovascular disease linked to alcohol intoxication. PUBLIC HEALTH RELEVANCE: Binge drinking is the predominant form of alcohol abuse in the US. Binge drinking may lead to cerebral artery constriction, vasospasm and stroke, all consequences of ethanol- induced contraction of cerebral artery smooth muscle. We identified that this ethanol action is mediated by smooth muscle ion channels of the BK type, which are complex heterooligomeric proteins. This proposal will identify the protein subunit site and molecular mechanism leading to ethanol inhibition of BK current and cerebral artery constriction. The proposal will bring critical information to design rational pharmacotherapeutic interventions in cerebrovascular disease associated with alcohol intoxication.