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: 10413935
• 负责人: Anna Bukiya
• 金额: $ 56.81万
• 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/10413935
• 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; 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.

人类和动物的数据表明，胆固醇（胆固醇）可能会破坏平滑肌（SM）的张力， 在没有任何动脉硬化或解剖异常迹象的情况下， 导致中风血管成分的作用。在更进一步的研究中， 脑动脉硬化的发病率越高，脑血管病 动脉平滑肌张力与内皮、微循环、炎症及其他循环因素有关。 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， 通过调节钙和电压门控来控制阻力大小、脑动脉SM张力和直径 大电导钾通道（BK）位于脑动脉SM质膜。这里，BK函数 主要由通道形成α（cbv 1）和调节β1亚基的偶联引起。一致行动 cbv 1 +β1的激活和最终的BK激活产生了与去极化相反的外向钾电流- 介导的钙内流限制SM收缩并有利于动脉扩张。根据初步数据，我们预测 在低β1表达下，与选择性受体的相互作用（通过直接结合和变构调节）， 在cbv 1胞质结构域中鉴定的胆固醇识别氨基酸共有基序（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 β1水平，CLR驱动的β1和cbv 1-β1功能偶联的质膜部分的增加将占上风， 导致BK电流增加、SM松弛和动脉扩张。我们预测，β1基因不同的脑动脉 表达将显示出不同的易感性，SM中的β-内酰胺酶水平将决定 β1依赖性血管扩张剂。我们的预测将沿着沿着三个具体目标（SA）进行检验。SA 1（分子水平）： 确定导致CLR诱导的BK（cbv 1）阻滞的结构基础和门控机制 同源四聚体）通过CLR-cbv 1 CRAC相互作用发挥功能。SA 2（细胞水平）：确定机制 这是cbv 1 +β1通道激活的基础。来自SA 1和SA 2的知识将被整合到SA 3（器官）中 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和动脉直径。 我们将联合收割机在计算建模、结合分析、质谱、微分 扫描荧光法，膜片钳和脂双层电生理学，变构门控分析，电穿孔 SM细胞和脑动脉的基因工程小鼠突变的cDNA，亚基运输，共聚焦成像 和脑动脉直径测定，确保可行性。我们希望挑战的范式， BK的调节是继发于双层物理化学性质的非特异性扰动，并提供 关于脑动脉功能控制的里程碑信息，这对于设计小型药物是必要的 可以将BK通道功能调节至可变的CLR水平并对抗TLR相关的脑血管疾病。