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

基本信息

批准号：
10627854
负责人：
Anna Bukiya
金额：
$ 59.47万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
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/10627854
关键词：
Address Amino Acids Animal Model Anterior Arteries Arteriosclerosis Atherosclerosis Binding Biological Assay Blood Vessels Brain Brain region Calcium Cell membrane Cell model Cells Cerebrovascular Circulation Cerebrovascular Disorders Cerebrum Chemosensitization Cholesterol Cognitive deficits Complementary DNA Computer Models Consensus Coupling Data Deformity Dementia Diameter Disease Electrophysiology (science)Electroporation Endothelium Engineering Ensure Functional disorder High Prevalence In Vitro Incidence Inflammatory Ion Channel Irrigation 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 Penetration Pharmaceutical Preparations Phenotype Physiological Physiology Potassium Potassium Channel Property Proteins Publishing Red Cross Regulation Resistance Risk Factors Role Sarcoplasmic Reticulum Scanning Secondary to Smooth Muscle Smooth Muscle Myocytes Stroke Tail Testing Therapeutic Agents Tissues Vasodilation Vasodilator Agents Voltage-Gated Potassium Channel animal data base cerebral artery cholesterol control confocal imaging constriction design efficacy evaluation hemodynamics human data in vivo intracranial artery large-conductance calcium-activated potassium channels middle cerebral artery novel parenchymal arterioles patch clamp pharmacologic stroke patient trafficking vascular factor voltage

项目摘要

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.

人类和动物的数据表明，胆固醇（CLR）可能会破坏平滑肌（SM）音调在没有任何动脉硬化或解剖异常的情况下，电阻大小的大脑动脉，该CLR 促成中风的血管成分的作用。进一步的一步，中风的患者大大脑动脉和高CLR将呈现较高的动脉硬化率。 CLR对大脑的作用动脉SM张力归因于内皮，微循环，炎症和其他循环因子。偏离了目前的范式，并得到了初步数据的支持，我们的中心假设是CLR可能控制电阻大小，大脑动脉SM张力和直径，通过调节钙和电压门控大电导（BK）的钾通道位于大脑动脉SM平原上。在这里，BK功能基本形成α（CBV1）和调节β1亚基的偶联的主要结果。一致的动作 CBV1+β1和最终BK激活会产生外向钾电流，与沉积相反介导的钙影响，限制SM收缩并有利于动脉词典。根据初步数据，我们预测在低β1表达下，CLR相互作用（通过直接结合和变构调制）与选定胆固醇识别氨基酸共识基序（CRAC）在CBV1胞质结构域中将领导 CLR诱导的BK电流减少，SM收缩和脑动脉收缩。相反，在高处 β1水平，CLR驱动的β1和CBV1-β1功能耦合的浆膜分数的增加将占上风导致BK电流增加，SM松弛和动脉词典。我们预测β1不同的脑动脉表达将显示出对CLR的差异脆弱性，SM中的CLR水平将调节 β1依赖性血管扩张剂。我们的预测将沿三个特定目标（SA）进行测试。 SA1（分子水平）：确定导致CLR诱导的BK阻碍的结构碱基和门控机制（CBV1 通过CLR-CBV1 CRAC相互作用函数均匀）。 SA2（细胞水平）：确定机制 CBV1+β1通道的CLR激活是基础。 SA1和SA2的知识将集成在SA3中（器官级别）：确定CLR-BK亚基相互作用对脑BK功能的后果使用体外和体内CLR递送方法在物理条件下的动脉SM和动脉直径。我们结合了计算建模，结合测定，质谱，差异的独特专业知识扫描荧光法，斑块钳和脂质双层电生理学，变构门控分析，电穿孔具有突变的cDNA，亚基运输，共聚焦成像的工程小鼠的SM细胞和大脑动脉和脑动脉直径的测定，确保可行性。我们希望挑战CLR的范式 BK的调节是双层物理化学特性的非特异性扰动的继发的，并提供关于脑动脉功能的CLR控制的里程碑信息，这对于设计小药物是必需的将BK通道功能调整为可变的CLR水平，并抵消与CLR相关的脑血管疾病。