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）： 间歇性的中度至重度酒精摄入，例如酗酒，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) 通道的活动严重限制血管肌细胞收缩，有利于脑血管扩张。中度间歇性饮酒后，循环中达到的乙醇水平（50 mM）会通过其 21 个亚基抑制脑动脉肌细胞 BK 通道的活性，导致不依赖于内皮的脑血管收缩。 除乙醇外，肌细胞膜中的高胆固醇也会抑制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。基于人工脂质双层的初步发现，我们假设 BK 21 亚基促进乙醇诱导的 BK 通道抑制的胆固醇放大，从而促进血管收缩。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从野生型（C57BL/6）和 KCNMB1 敲除小鼠脑动脉中新鲜分离的肌细胞中 BK 通道活性的降低。虽然野生型动物的动脉肌细胞 BK 通道具有功能性 21 个亚基，但 KCNMB1 敲除型动物缺乏该亚基。实验将使用细胞膜中的完整胆固醇、以及胆固醇耗尽后和胆固醇富集进行。在上述条件下用膜片钳记录 BK 电流将使我们能够比较乙醇-胆固醇对 BK 通道的协同抑制程度与没有 21 个亚基的情况。我们期望证明，BK 21 亚基可作为胆固醇促进剂，放大乙醇诱导的动脉肌细胞 BK 通道抑制。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）通过抑制血管中含有 21 的 BK 通道来损害脑动脉扩张。平滑肌本身。 公众健康相关性：酒精和胆固醇是中风和脑动脉松弛受损的两个众所周知的危险因素。我们的目的是证明 BK 通道 21 亚基通过抑制含有 21 亚基的 BK 通道，促进酒精引起的脑血管痉挛的胆固醇放大。这项研究将支持酒精研究的新概念，并将为酒精性脑血管疾病的治疗干预开辟新的场所。