Ethanol actions on slo channels from arteries vs. brain

描述（由申请人提供）：已知脑动脉急性暴露于酒精（如酗酒）可引起脑血管痉挛和中风。这种病理的风险因损害脑血管舒张的机制（如高胆固醇）而增加，大多数证据表明，酒精诱导的脑动脉收缩是由于乙醇（EtOH）直接收缩脑平滑肌。然而，确定etoh诱导的脑血管收缩的离子机制尚不清楚。大电导，钙离子激活的脑动脉平滑肌的K + (BK)通道活性严重限制了收缩程度。BK通道由α亚基（由slo基因编码）和β亚基组成。Slo表达呈现的电流具有BK通道的所有关键特征。本应用程序的最终目的是确定EtOH调节脑血管BK通道活性的分子因素和机制，以及这种调节对急性EtOH诱导的脑血管平滑肌收缩的贡献。基于我们之前的数据，我们已经缩小了决定特定BK通道对急性EtOH暴露反应的分子实体：1)特定的慢通道异构体；2)慢酶亚基的脂质（特别是类固醇）环境。因此，我们将采用药理学、电生理学（膜片钳）和分子生物学技术相结合的方法，通过顺序检查药物在以下方面的作用，全面研究EtOH对脑血管平滑肌BK通道功能的调节：1)分离的脑动脉，确定EtOH对BK通道功能的调节对酒精对脑动脉收缩作用的贡献；2)分离的脑血管肌细胞，当通道嵌入其天然膜环境时，研究直接的EtOH-BK通道复合物相互作用；利用我们最近从脑动脉肌细胞中克隆的slo，建立模型系统，探测直接的slo亚基- etoh相互作用，并建立其分子决定因素，包括膜类固醇的调节。明确EtOH作用于脑血管BK通道的分子机制将为酒精性脑血管疾病潜在治疗药物的基本原理设计提供基础信息。

DESCRIPTION (provided by applicant): Acute exposure of cerebral arteries to alcohol (as in binge drinking) is known to cause cerebral vasospasm and stroke. The risk for this pathology is increased by mechanisms that impair cerebral vasodilation, such as high cholesterol Most of the evidence indicates that alcohol-induced cerebral artery constriction is due to a direct contraction of cerebral smooth muscle by ethanol (EtOH). However, the ionic mechanisms that determine EtOH-induced cerebrovascular contraction are unknown. Large conductance, Ca++-activated K + (BK) channel activity in the cerebral artery smooth muscle critically limits the degree of contraction. BK channels consist of alpha (encoded by the slo gene) and beta subunits. Slo expression renders currents that bear all the key features of BK channels. The ultimate goal of this application is to identify the molecular factors and mechanisms determining EtOH modulation of cerebrovascular BK channel activity and the contribution of this modulation to cerebrovascular smooth muscle contraction induced by acute EtOH. Based on our previous data, we have narrowed down the molecular entities determining a particular BK channel response to acute EtOH exposure to: 1) the particular slo channel isoform; and 2) the lipid (in particular, steroid) environment of the slo subunit. Therefore, using a combination of pharmacological, electrophysiological (patch-clamp) and Molecular Biology techniques, here we will comprehensively address EtOH modulation of BK channel function in cerebrovascular smooth muscle by sequentially examining drug action in: 1) the isolated cerebral artery to determine the contribution of EtOH modulation of BK channel function to alcohol action on cerebral artery constriction; 2) isolated cerebrovascular myocytes to address a direct EtOH-BK channel complex interaction when the channel is embedded in its natural membrane environment and, taken advantage of our recent cloning of slo from cerebral artery myocytes, 3) model systems to probe a direct slo subunit-EtOH interaction and establish its molecular determinants, including modulation by membrane steroids. Pinpointing the molecular mechanisms involved in EtOH action on cerebrovascular BK channels will bring fundamental information for the rationale design of drugs of potential therapeutic use in alcohol-induced cerebrovascular disease.