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.

人类和动物数据表明，胆固醇 (CLR) 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，阻力大小的脑动脉，这种 CLR 更进一步，中风患者的功能障碍会导致中风的血管成分。 脑动脉和高 CLR 会导致 CLR 对脑动脉硬化的发生率较高。 动脉SM张力可归因于内皮、微循环、炎症和其他循环因素。 与当前的范式不同并得到初步数据的支持，我们的中心假设是 CLR 可能 通过调节钙门控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) 位于大脑动脉 SM 质膜中，BK 发挥作用。 主要是通道形成α (cbv1) 和调节β1 亚基耦合的协同作用。 cbv1+β1 和最终的 BK 激活产生向外的钾电流，反对去极化- 介导钙内流，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，CLR 与选定的相互作用（通过直接结合和变构调节） 胆固醇识别 cbv1 胞质结构域中鉴定的氨基酸共有基序 (CRAC) 将导致 相比之下，CLR 引起的 BK 电流减少，SM 收缩和脑动脉收缩。 β1 水平、CLR 驱动的 β1 质膜分数增加和 cbv1-β1 功能耦合将占上风， 导致 BK 电流增加、SM 松弛和动脉扩张，我们预测脑动脉的 β1 有所不同。 SM 中的 CLR 水平将决定其功效 我们的预测将沿着三个特定目标（SA1）进行测试： 确定导致 CLR 诱导的 BK 阻碍的结构基础和门控机制 (cbv1 同四聚体）通过 CLR-cbv1 CRAC 相互作用发挥作用：确定机制。 SA1 和 SA2 的 CLR 激活基础知识将整合到 SA3（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 使用体外和体内 CLR 递送方法在生理条件下对动脉 SM 和动脉直径进行影响。 我们结合了计算建模、结合测定、质谱、微分分析等方面的独特专业知识 扫描荧光测定法、膜片钳和脂质双层电生理学、变构门控分析、电穿孔 来自具有突变 cDNA 的工程小鼠的 SM 细胞和脑动脉、亚基运输、共聚焦成像 我们期望挑战确保 CLR 的范式。 BK 的调节是双层物理化学性质的非特异性扰动的次要因素，并且提供 CLR 控制脑动脉功能的里程碑信息，这对于设计小药物来说是必要的 调节 BK 通道功能以适应不同的 CLR 水平并抵消 CLR 相关的脑血管疾病。