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，代表了美国和其他工业化国家酒精消费的主要形式。酒精滥用是中风患者最常见的三个危险因素之一，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、钙和电压门控（BK）通道的活性严重限制了血管肌细胞的收缩，有利于脑血管扩张。中度重度间歇饮酒（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，导致内皮不依赖型脑血管收缩。除乙醇外，肌细胞膜中的高胆固醇水平也会抑制BK电流，降低血管顺应性，有利于血管收缩。胆固醇被认为会放大乙醇驱动的BK通道抑制，因此，乙醇和高胆固醇水平会对血管顺应性产生深远的负面影响。基于人工脂质双分子层的初步发现，我们假设bk21亚基促进了乙醇诱导的BK通道抑制的胆固醇扩增，从而导致血管收缩。在本研究中，我们的目标是使用膜片钳电生理学来记录乙醇诱导的野生型（C57BL/6）和KCNMB1敲除小鼠脑动脉中新鲜分离的肌细胞BK通道活性的降低。野生型动物的动脉肌细胞BK通道具有21个功能性亚基，而KCNMB1敲除缺乏这个亚基。实验将在细胞膜中完整的胆固醇，以及耗尽胆固醇和胆固醇富集后进行。在上述条件下膜片钳记录BK电流将使我们能够比较乙醇-胆固醇对BK通道的协同抑制程度，有或没有21个亚基。我们期望证明，bk21亚基作为胆固醇的促进剂，可以放大乙醇诱导的动脉肌细胞BK通道的抑制。在分离的肌细胞上获得的结果将与野生型和KCNMB1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和胆固醇（两个众所周知的中风危险因素）后循环中的乙醇浓度通过抑制血管平滑肌中含有21的BK通道而损害脑动脉扩张。

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.