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.

大脑需要大量循环 氧气和营养。电阻大小的大脑动脉通过肌原性控制恒定的血液流向大脑 自动调节机制。异常胆固醇水平触发抗性大小的大脑失调 大型电导（BK）的钙和电压门控钾通道的动脉，有助于 常见的脑血管病理（例如中风），认知定义了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通道的胆固醇抑制变化 血管平滑肌影响大脑动脉直径的收缩力，并失调氧气的输送 和整个大脑的营养。而胆固醇会减少BK通道活性，而分子 当前发生这种情况的机制尚不清楚。胆固醇识别/相互作用氨基酸 共识（CRAC）基序是胆固醇的潜在结合位点，在整个BK通道中发现了十个 氨基酸序列。胞质尾域包含这十个crac图案中的七个，它已经 证明胆固醇通过其中一个或多个这些胞质尾域CRAC图案调节BK电流。 我的目标是确定由BK通道调节胆固醇调节的分子机制 与某些胞质尾域CRAC基序相互作用，并定义该调节对脑的影响 动脉直径。该提案针对两个主要目的：AIM 1将确定结构性基础和门控 通过胆固醇直接相互作用与胆固醇引起的BK功能的阻碍的机制 BK通道形成的SLO1亚基。胆固醇通过与 特定的CRAC将通过电生理学和结合实验来解决。我还将确定哪个BK 胆固醇相互作用时，门控参数会改变。 1.1。我将首先确定独特的贡献 CRAC对胆固醇结合的基序以及随之而来的同源性SLO1通道活性的抑制作用。 1.2。下一个， 我将确定不同的CRAC图案的关键物理特征，以调制 频道的胆固醇敏感性。 1.3。最后，我将确定引导的胆固醇敏感的门控参数 胆固醇引起的阻碍SLO1通道活性。 AIM 2将解决生理和 胆固醇-SLO1通过CRAC4基序相互作用的药理后果作为天然BKS的一个例子 在脑动脉平滑肌和脑动脉直径中。 2.1。我将确定胆固醇的影响 在生理条件下，在动脉肌细胞中与本机BK中的CRAC4相互作用。 2.2。我也会 通过SLO1 CRAC4在动脉直径上确定BK电流调节胆固醇的后果。这 提案将首次制定统一计划，该计划解释胆固醇在BK频道上的行动 基于立体声的直接结合，在分子和细胞水平上的功能和脑动脉直径 到BK通道形成的SLO1亚基和/或固醇相互作用继发的变构调节。