Cholesterol regulation of smooth muscle BK channel proteins and consequent control of cerebral artery diameter

胆固醇对平滑肌 BK 通道蛋白的调节以及随后对脑动脉直径的控制

• 批准号: 10063416
• 负责人: Anna Bukiya
• 金额: $ 64.6万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063416
• 关键词: Address; Amino Acids; Animal Model; Anterior; Arteries; Arteriosclerosis; Atherosclerosis; Binding; Biological Assay; Blood Vessels; Brain; Brain region; Calcium; Caliber; Cell membrane; Cell model; Cells; Cerebrovascular Circulation; Cerebrovascular Disorders; Cerebrum; Chemosensitization; Cholesterol; Cognitive deficits; Complementary DNA; Computer Models; Consensus; Coupling; Data; Deformity; Dementia; Disease; Electrophysiology (science); Electroporation; Endothelium; Engineering; Ensure; Functional disorder; High Prevalence; In Vitro; Incidence; Inflammatory; Ion Channel; Knowledge; Lead; Link; Lipid Bilayers; Lipids; Mass Spectrum Analysis; Mediating; Methods; Microcirculation; Microvascular Dysfunction; Modification; Molecular; Morphology; Mus; Muscle Contraction; Muscle Tonus; Muscle relaxation phase; Mutate; Organ; Pathologic; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Phenotype; Physiological; Physiology; Potassium; Potassium Channel; Property; Proteins; Red Cross; Regulation; Resistance; Risk Factors; Role; Sarcoplasmic Reticulum; Scanning; Secondary to; Smooth Muscle; Smooth Muscle Myocytes; Stroke; Structure; Tail; Testing; Therapeutic Agents; Tissues; Vasodilation; Vasodilator Agents; Voltage-Gated Potassium Channel; animal data; base; cerebral artery; cholesterol control; confocal imaging; constriction; design; hemodynamics; human data; in vivo; intracranial artery; large-conductance calcium-activated potassium channels; middle cerebral artery; novel; parenchymal arterioles; patch clamp; stroke patient; trafficking; vascular factor; voltage

Human and animal data demonstrate that cholesterol (CLR) may disrupt the smooth muscle (SM) tone of resistance-size cerebral arteries in absence of any sign of arteriosclerosis or anatomical abnormalities, this CLR action contributing to a vascular component of stroke. In a further step, stroke patients with dysfunction of large cerebral arteries and high CLR will present a higher prevalence of arteriosclerosis. CLR actions on cerebral artery SM tone have been attributed to endothelial, microcirculation, inflammatory and other circulating factors. Departing from this current paradigm and supported by preliminary data, our central hypothesis is that CLR may control resistance-size, cerebral artery SM tone and diameter via regulation of calcium- and voltage-gated potassium channels of big conductance (BK) located in the cerebral artery SM plasmalemma. Here, BK function primarily results from the coupling of channel-forming α (cbv1) and regulatory β1 subunits. The concerted action of cbv1+β1 and eventual BK activation generates outward potassium current that opposes depolarization- mediated calcium influx, limits SM contraction and favors artery dilation. Based on preliminary data, we predict that under low β1 expression, CLR interaction (through direct binding and allosteric modulation) with selected Cholesterol Recognition Amino acid Consensus motifs (CRACs) identified in the cbv1 cytosolic domain will lead to CLR-induced reduction of BK current, SM contraction and cerebral artery constriction. In contrast, under high β1 levels, CLR-driven increases in the plasmalemmal fraction of β1 and cbv1-β1 functional coupling will prevail, leading to increased BK current, SM relaxation and artery dilation. We predict that brain arteries that differ in β1 expression will display a differential vulnerability to CLR, and that CLR levels in SM will condition the efficacy of β1-dependent vasodilators. Our predictions will be tested along three specific aims (SA). SA1 (molecular level): determine the structural bases and gating mechanisms that lead to CLR-induced hindering of BK (cbv1 homotetramer) function through CLR-cbv1 CRAC interactions. SA2 (cellular level): determine the mechanisms that underlie CLR activation of cbv1+β1 channels. Knowledge from SA1 and SA2 will be integrated in SA3 (organ level): determine the consequences of CLR-BK subunit interactions on the function of native BKs in cerebral artery SM and on artery diameter under physiological conditions using in vitro and in vivo CLR delivery methods. We combine unique expertise in computational modeling, binding assays, mass spectroscopy, differential scanning fluorimetry, patch-clamp and lipid bilayer electrophysiology, allosteric gating analysis, electroporation of SM cells and cerebral arteries from engineered mice with mutated cDNAs, subunit trafficking, confocal imaging and cerebral artery diameter determinations, ensuring feasibility. We expect to challenge the paradigm that CLR modulation of BK is secondary to nonspecific perturbation of bilayer physico-chemical properties, and to provide milestone information on CLR control of cerebral artery function, which will be necessary to design small drugs that adjust BK channel function to variable CLR levels and counteract CLR-associated cerebrovascular disease.

人类和动物数据表明，胆固醇(CLR)可能会扰乱血管平滑肌(SM)的张力 阻力大小的脑动脉，没有任何动脉硬化或解剖异常的迹象，这项CLR 导致中风的血管成分的动作。在进一步的步骤中，中风患者功能障碍较大 脑动脉和高CLR将呈现更高的动脉硬化患病率。CLR对大脑的作用 动脉Sm张力与内皮、微循环、炎症等循环因素有关。 从目前的范式出发，并得到初步数据的支持，我们的中心假设是，CLR可能 通过调节钙和电压门控来控制阻力-大小、脑动脉Sm音调和直径 大电导钾通道(BK)位于大脑动脉SM质膜。这里，BK函数 主要由通道形成α(CBV1)和调节β1亚基的偶联所致。协同行动 Cbv1+β1和最终的BK激活产生与去极化相反的外向钾电流- 调节钙内流，限制SM收缩，有利于动脉扩张。根据初步数据，我们预测 在β-1低表达情况下，CLR通过直接结合和变构调节与 在cbv1胞浆结构域中发现的胆固醇识别氨基酸共识基序(CRAC)将导致 对CLR引起的BK电流、SM收缩和脑动脉收缩的抑制作用。相比之下，在高 β-1水平，CLR驱动的β-1和CBV1-β-1功能偶联的血浆成分增加将占优势， 导致BK电流增加、SM松弛和动脉扩张。我们预测在β1中不同的脑动脉 表达式将显示CLR的不同漏洞，SM中的CLR水平将决定 β-1依赖的血管扩张剂。我们的预测将根据三个具体目标(SA)进行测试。SA1(分子水平)： 确定导致CLR诱导的BK(Cbv1)障碍的结构基础和门控机制 同源四聚体)通过CLR-cbv1 CRAC相互作用发挥作用。SA2(细胞水平)：确定机制 这是CBV1+β1通道CLR激活的基础。来自SA1和SA2的知识将被整合到SA3(器官 水平)：确定CLR-BK亚基相互作用对大脑中天然BK功能的影响 动脉SM和生理条件下对动脉内径的影响采用体外和体内CLR给药方法。 我们结合了计算建模、结合分析、质谱学、差示等方面的独特专业知识 扫描荧光法、膜片钳和脂质双层电生理学、变构门控分析、电穿孔 基因工程小鼠的SM细胞和脑动脉的突变、亚单位运输、共聚焦成像 和大脑动脉直径的测定，以确保可行性。我们希望挑战CLR BK的调制次于双层物理化学性质的非特异性扰动，并提供 CLR控制大脑动脉功能的里程碑信息，这将是设计小型药物所必需的 调节BK通道功能以适应不同的CLR水平，并对抗CLR相关性脑血管疾病。