Ethanol actions on slo channels from arteries vs. brain

描述(由申请人提供)：众所周知，大脑动脉急性接触酒精(如酗酒)会导致脑血管痉挛和中风。这种病理的风险是由损害脑血管扩张的机制增加的，例如高胆固醇。大多数证据表明，酒精引起的脑动脉收缩是由于乙醇直接收缩大脑平滑肌(Etoh)。然而，决定乙醇引起脑血管收缩的离子机制尚不清楚。大电导、钙激活的K+(BK)通道活动在大脑动脉平滑肌中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表达式呈现具有BK通道的所有关键特征的电流。本研究的最终目的是确定乙醇调节脑血管BK通道活性的分子因素和机制，以及这种调节在急性乙醇引起的脑血管平滑肌收缩中的作用。基于我们以前的数据，我们已经缩小了决定特定BK通道对急性乙醇暴露的反应的分子实体：1)特定的SLO通道亚型；以及2)SLO亚基的脂质(特别是类固醇)环境。因此，我们将结合药理学、电生理学(膜片钳)和分子生物学技术，通过依次检测下列药物的作用来全面研究乙醇对脑血管平滑肌BK通道功能的调节：1)确定乙醇对BK通道功能的调节在酒精对脑动脉收缩作用中的贡献；2)分离脑血管心肌细胞，以研究当通道嵌入其自然膜环境中时，EtoH-BK通道复合体的直接相互作用；并利用我们最近从大脑动脉肌细胞克隆的SLO，3)模型系统，探索SLO亚单位与Etoh的直接相互作用，并建立其分子决定因素，包括膜类固醇的调节。弄清乙醇对脑血管BK通道作用的分子机制，将为设计治疗酒精性脑血管疾病的潜在药物提供基础信息。

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.