Cholesterol modulation of BK currents and cerebral artery diameter via channel-forming slo1 subunits

胆固醇通过通道形成 slo1 亚基调节 BK 电流和脑动脉直径

• 批准号: 10751934
• 负责人: Elizabeth Hope Schneider
• 金额: $ 3.88万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751934
• 关键词: Address; Adult; Alcohols; Allosteric Regulation; Amino Acid Sequence; Amino Acids; Arteries; Atherosclerosis; Binding; Binding Sites; Biochemical; Biological Assay; Blood Vessels; Blood flow; Brain; Calcium; Cellular Membrane; Cerebrovascular Circulation; Cerebrum; Cholesterol; Cognitive deficits; Consensus; Data; Dementia; Dialysis procedure; Diameter; Electrophysiology (science); Equilibrium; Functional disorder; Goals; Homeostasis; Individual; Knock-in Mouse; Knock-out; Knowledge; Life; Ligands; Modernization; Modification; Molecular; Monitor; Muscle Cells; Muscle Tonus; Nutrient; Oxygen; Pathologic; Pathology; Pattern; Physiological; Physiology; Point Mutation; Protein Family; Protein Isoforms; Proteins; Publishing; Recreation; Regulation; Resistance; Scanning; Scheme; Secondary to; Site-Directed Mutagenesis; Smooth Muscle; Sterols; Stroke; Systemic blood pressure; Tail; Testing; Time; Tyrosine; United States; Vascular Smooth Muscle; Voltage-Gated Potassium Channel; cerebral artery; cerebrovascular; cerebrovascular pathology; drug of abuse; experimental study; hypercholesterolemia; large-conductance calcium-activated potassium channels; mouse model; mutant; nanoscale; patch clamp; pharmacologic; rational design; receptor; response; western diet

Regulation of cerebral blood flow is necessary for survival as the brain requires a large amount of circulating oxygen and nutrients. Resistance-size cerebral arteries manage constant blood flow to the brain by myogenic autoregulation mechanisms. Abnormal cholesterol levels trigger dysregulation of resistance-size cerebral arteries via the calcium- and voltage-gated potassium channel of large conductance (BK), contributing to common cerebrovascular pathologies such as stroke, cognitive deficits including some forms of dementia, and the disruption of cerebral artery function by recreational alcohol. Cholesterol inhibition of the BK channel alters contractility of vascular smooth muscle impacting cerebral artery diameter, and dysregulates delivery of oxygen and nutrients throughout the brain. While cholesterol diminishes BK channel activity, the molecular mechanism(s) by which this occurs are currently unknown. Cholesterol recognition/interaction amino acid consensus (CRAC) motifs are potential binding sites for cholesterol, and ten are found throughout the BK channel amino acid sequence. The cytosolic tail domain contains seven of these ten CRAC motifs, and it has been demonstrated that cholesterol modulates BK currents by one or more of these cytosolic tail domain CRAC motifs. My goal is to determine the molecular mechanisms that govern cholesterol regulation of the BK channel by interacting with certain cytosolic tail domain CRAC motif(s), and to define the impact of this regulation on cerebral artery diameter. This proposal addresses two main aims: Aim 1 will determine the structural basis and gating mechanisms that lead to cholesterol-induced hindering of BK function through cholesterol direct interactions with the BK channel-forming slo1 subunit. The hypothesis that cholesterol modulates BK currents via interaction with specific CRACs will be addressed by electrophysiology and binding experiments. I will also identify which BK gating parameter(s) are altered upon cholesterol interaction. 1.1. I will first determine the contribution of distinct CRAC motifs to cholesterol binding and the consequent inhibition of homomeric slo1 channel activity. 1.2. Next, I will determine the critical physicochemical features of distinct CRAC motifs that allow for modulation of the channel’s cholesterol sensitivity. 1.3. Finally, I will identify the cholesterol-sensitive gating parameters that lead to cholesterol-induced hindering of slo1 channel activity. Aim 2 will address the physiological and pharmacological consequences of cholesterol-slo1 interactions via CRAC4 motif as an example on native BKs in cerebral artery smooth muscle and cerebral artery diameter. 2.1. I will determine the effects of cholesterol interactions with CRAC4 in native BKs in arterial myocytes under physiological conditions. 2.2. I will also determine the consequences of cholesterol regulation of BK currents via slo1 CRAC4 on artery diameter. This proposal will for the first time develop a unifying scheme that explains the actions of cholesterol on BK channel function and cerebral artery diameter at both molecular and cellular levels based on direct binding of the sterol to the BK channel-forming slo1 subunit and/or allosteric regulation secondary to the sterol interaction.

脑血流量的调节对于生存是必要的，因为大脑需要大量的循环 氧气和营养物质。阻力大小的脑动脉通过肌源性管理流向大脑的持续血流 自动调节机制。异常的胆固醇水平会引发抵抗力大小的大脑失调 动脉通过大电导 (BK) 的钙门控和电压门控钾通道，有助于 常见的脑血管疾病，例如中风、认知缺陷（包括某些形式的痴呆症）和 娱乐性酒精对脑动脉功能的破坏。 BK 通道的胆固醇抑制改变 血管平滑肌的收缩性影响脑动脉直径，并调节氧气输送 以及整个大脑的营养。虽然胆固醇会降低 BK 通道活性，但分子 目前尚不清楚发生这种情况的机制。胆固醇识别/相互作用氨基酸 共有 (CRAC) 基序是胆固醇的潜在结合位点，在整个 BK 通道中发现了 10 个 氨基酸序列。胞质尾部结构域包含这十个 CRAC 基序中的七个，并且已被 证明胆固醇通过这些胞质尾部结构域 CRAC 基序中的一个或多个调节 BK 电流。 我的目标是确定控制 BK 通道胆固醇调节的分子机制 与某些胞质尾部结构域 CRAC 基序相互作用，并确定这种调节对大脑的影响 动脉直径。该提案解决了两个主要目标：目标 1 将确定结构基础和门控 通过胆固醇与 BK 的直接相互作用，导致胆固醇诱导 BK 功能受阻的机制 BK 通道形成 slo1 亚基。胆固醇通过与 BK 相互作用调节 BK 电流的假设 特定的 CRAC 将通过电生理学和结合实验来解决。我也会确定哪个BK 门控参数因胆固醇相互作用而改变。 1.1.我将首先确定不同的贡献 CRAC 基序与胆固醇结合并随后抑制同源 slo1 通道活性。 1.2.下一个， 我将确定不同 CRAC 基序的关键物理化学特征，这些特征允许调节 通道的胆固醇敏感性。 1.3.最后，我将确定导致胆固醇敏感的门控参数 胆固醇诱导的 slo1 通道活性阻碍。目标 2 将解决生理和 以天然 BK 为例，胆固醇与 slo1 通过 CRAC4 基序相互作用的药理学后果 脑动脉平滑肌和脑动脉直径。 2.1.我将确定胆固醇的影响 生理条件下动脉肌细胞中天然 BK 与 CRAC4 的相互作用。 2.2.我也会 确定通过 slo1 CRAC4 调节 BK 电流的胆固醇对动脉直径的影响。这 该提案将首次制定一个统一方案来解释胆固醇对 BK 通道的作用 基于甾醇的直接结合在分子和细胞水平上的功能和脑动脉直径 BK 通道形成 slo1 亚基和/或继发于甾醇相互作用的变构调节。