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

BK亚基在乙醇-胆固醇协同抑制BK通道中的作用

• 批准号: 8146995
• 负责人: Anna Bukiya
• 金额: $ 3.56万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-25 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8146995
• 关键词: Acute; Address; Alcohol abuse; Alcohol consumption; Alcoholic Beverages; Alcohols; Animals; Arteries; Blood Circulation; Blood Vessels; Calcium; Cell membrane; Cerebral Ischemia; Cerebrovascular Disorders; Cerebrovascular Spasm; Cessation of life; Cholesterol; Data; Developed Countries; Diet; Electrophysiology (science); Endothelium; Environment; Ethanol; Exposure to; Foundations; Functional disorder; Funding; Grant; Human; In Vitro; Ischemia; Ischemic Stroke; Knockout Mice; Lead; Lipid Bilayers; Mediating; Medical Research; Membrane; Modeling; Mus; Muscle Cells; Organ; Patients; Physiological; Potassium; Proteins; Rattus; Relaxation; Research Personnel; Risk; Risk Factors; Role; Smooth Muscle; Stroke; Therapeutic Intervention; Tissues; Titrations; Vascular Smooth Muscle; Vasodilation; alcohol research; base; binge drinking; cerebral artery; cerebrovascular; constriction; hypercholesterolemia; large-conductance calcium-activated potassium channels; novel; patch clamp; public health relevance; research study; synergism; vasoconstriction; voltage

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.

描述（由申请人提供）：偶发性，中度到体重的酒精摄入（例如暴饮暴食）代表了美国和其他工业化国家饮酒的主要形式。酗酒是中风患者中三个最常见的危险因素之一，暴饮暴食与脑血管缺血和中风的风险增加有关。脑血管缺血是由于血管舒张和/或增强脑动脉收缩而导致的。大传导，钙和电压门控（BK）通道的活性严重限制了血管肌细胞收缩，有利于脑血管扩张。中度重度饮酒（50 mM）后，循环水平达到的乙醇抑制了脑动脉心肌细胞BK通道的活性，从而通过其21个亚基抑制了内皮细胞独立的脑血管约束。 除乙醇外，心肌细胞膜中的高胆固醇水平也抑制了BK电流，减少血管依从性，有利于血管收缩。提议胆固醇扩增乙醇驱动的BK通道抑制作用，因此，乙醇和高胆固醇水平将对血管依从性产生深远的负面影响。基于人工脂质双层中的初步发现，我们假设BK 21亚基有助于乙醇诱导的BK通道抑制，从而促进胆固醇的扩增，从而促进血管收缩。在本研究中，我们旨在使用斑块钳电生理学来记录乙醇引起的从野生型（C57BL/6）和KCNMB1敲除小鼠脑动脉的新鲜分离的肌细胞中BK通道活性的降低。尽管野生型动物具有具有功能性21个亚基的动脉肌细胞BK通道，但KCNMB1敲除缺乏此亚基。实验将在细胞膜中以完整的胆固醇以及胆固醇耗尽和胆固醇富集后进行实验。在上述条件下对BK电流记录的斑块钳记录将使我们能够与无21个亚基的BK通道的乙醇 - 胆固醇协同抑制程度进行比较。我们预计会证明，BK 21亚基是胆固醇扩增乙醇诱导的动脉肌细胞BK通道的促进剂。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进行实验相匹配。 我们的研究将增强酒精研究和脑血管病理生理学的新概念：中度至重度发作性饮酒和胆固醇后在循环中获得的乙醇浓度，这是两种众所周知的中风风险因素，通过抑制血管平滑肌本身的21个含BK通道，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放松受损的两个众所周知的危险因素。我们的目的是表明，BK 21个亚基通过抑制21个含21个亚基的BK通道来促进酒精诱导的大脑血管痉挛的胆固醇扩增。这项研究将加强酒精研究中的新概念，并将为酒精驱动的脑血管疾病开设一个新的场所。