Role of BK subunits in ethanol-cholesterol synergistic inhibition of BK channel

描述（由申请人提供）：间歇性、中度至重度酒精摄入，如酗酒，是美国和其他工业化国家酒精消费的主要形式。酗酒是中风患者中三个最常见的危险因素之一，酗酒与脑血管缺血和中风的风险增加有关。脑血管缺血是由血管舒张受损和/或脑动脉收缩增强引起的。大电导、钙门控和电压门控（BK）通道的活性严格限制了血管肌细胞收缩，有利于脑血管扩张。中度-重度间歇性饮酒（50 mM）后循环中达到的乙醇水平通过其21个亚基抑制脑动脉肌细胞BK通道的活性，导致非内皮依赖性脑血管收缩。 除乙醇外，心肌细胞膜中高胆固醇水平也抑制BK电流，降低血管顺应性，有利于血管收缩。胆固醇被认为放大了乙醇驱动的BK通道抑制，因此，乙醇和高胆固醇水平将对血管顺应性产生深远的负面影响。基于人工脂质双层的初步研究结果，我们假设BK 21亚基促进胆固醇放大乙醇诱导的BK通道抑制，从而，血管收缩。在本研究中，我们的目的是使用膜片钳电生理学记录乙醇诱导的BK通道活性的下降，新鲜分离的野生型（C57 BL/6）和KCNMB 1基因敲除小鼠脑动脉的心肌细胞。野生型动物动脉肌细胞BK通道具有21个功能亚基，而KCNMB 1敲除缺乏该亚基。实验将在细胞膜中完整的胆固醇以及胆固醇耗尽和胆固醇富集后进行。在上述条件下用膜片钳记录BK电流，将使我们能够比较有或没有21亚基时乙醇-胆固醇协同抑制BK通道的程度。我们期望证明，BK 21亚基作为胆固醇放大乙醇诱导的动脉肌细胞BK通道抑制的促进剂。在分离的肌细胞上获得的结果将与来自野生型和KCNMB 1敲除小鼠的体外加压脑动脉实验相匹配。 我们的研究将支持酒精研究和脑血管病理生理学中的一个新概念：中度至重度间歇性饮酒后循环中获得的乙醇浓度和胆固醇，两个众所周知的中风危险因素，通过抑制血管平滑肌本身中含有21的BK通道来损害脑动脉扩张。 公共卫生相关性：酒精和胆固醇是中风和脑动脉舒张受损的两个众所周知的危险因素。我们的目的是表明BK通道21亚基通过抑制含有21亚基的BK通道促进酒精诱导的脑血管痉挛的胆固醇放大。这项研究将为酒精研究提供一个新的概念，并将为酒精驱动的脑血管疾病的治疗干预开辟一个新的途径。

DESCRIPTION (provided by applicant): Episodic, moderate-to-heavy alcohol intake, such as in binge drinking, represents the primary form of alcohol consumption in the US and other industrialized countries. Alcohol abuse is one of the three most common risk factors among stroke patients, with binge drinking being associated with an increased risk for cerebrovascular ischemia and stroke. Cerebrovascular ischemia results from impaired vasodilation and/or enhanced constriction of cerebral arteries. The activity of large-conductance, calcium- and voltage-gated (BK) channels critically limits vascular myocyte contraction, favoring cerebrovascular dilation. Ethanol at levels reached in circulation after moderate-heavy episodic drinking (50 mM) inhibits the activity of cerebral artery myocyte BK channels via their21 subunits, leading to endothelium-independent cerebrovascular constriction. Apart from ethanol, high cholesterol levels in myocyte membrane also inhibit BK currents, decrease vascular compliance, favoring vasoconstriction. Cholesterol is proposed to amplify ethanol-driven BK channel inhibition, thus, ethanol and high cholesterol level would have a profound negative impact on vascular compliance. Based on preliminary findings in artificial lipid bilayers, we hypothesize that BK 21 subunit facilitates cholesterol amplification of ethanol-induced BK channel inhibition and thus, vasoconstriction. In the present study we are aiming to use patch-clamp electrophysiology to record ethanol-induced decrease in BK channel activity in myocytes freshly isolated from wild type (C57BL/6) and KCNMB1 knock-out mouse cerebral arteries. While wild type animals have arterial myocyte BK channels with functional 21 subunits, KCNMB1 knock-outs lack this subunit. Experiments will be performed with intact cholesterol in the cell membranes, as well as after depletion of cholesterol, and cholesterol enrichment. Patch-clamp recording of BK currents under conditions desrcribed above will allow us to compare degree of ethanol-cholesterol synergistic inhibition of BK channel with of without 21 subunits. We anticipate to prove, that BK 21 subunit serves as a facilitator for cholesterol to amplify ethanol-induced inhibition of arterial myocyte BK channel. Results obtained on isolated myocytes will be matched with experiments on in vitro pressurized cerebral arteries from wild type and KCNMB1 knock-out mice. Our studies will bolster a novel concept in alcohol research and cerebrovascular pathophysiology: ethanol concentrations obtained in circulation after moderate to heavy episodic drinking and cholesterol, two well-known risk factors for stroke, impair cerebral artery dilation by inhibiting 21-containing BK channels in vascular smooth muscle itself. PUBLIC HEALTH RELEVANCE: Alcohol and cholesterol are two well-known risk factors for stroke and impaired cerebral artery relaxation. We are aiming to show that BK channel 21 subunits facilitate cholesterol amplification of alcohol-induced cerebral vasospasm via inhibition of 21 subunit-containing BK channels. This study will bolster a novel concept in alcohol research and will open a new venue for therapeutic intervention on alcohol-driven cerebrovascular disease.