Role of BK Channel Across Alcohol Behaviors

BK 通道在酒精行为中的作用

基本信息

批准号：
9754725
负责人：
Angela Renee Ozburn
金额：
$ 6.3万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-05 至 2020-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9754725
关键词：
Action Potentials Acute Alcohol withdrawal syndrome Alcohols Behavior Behavioral Binding Sites Biological Assay Brain CRISPR/Cas technology Caenorhabditis elegans Calcium Calcium-Activated Potassium Channel Collaborations Complex Consumption Convulsions Dependence Dose Engineering Ethanol Ethanol dependence Exhibits Extracellular Matrix Proteins Genetic Screening Human Injections Intoxication Knock-in Knock-in Mouse Knock-out Knockout Mice Lipids Mammals Measurement Measures Mediating Membrane Metabolic Clearance Rate Metabolism Methods Modeling Molecular Molecular Conformation Molecular Target Motor Activity Mus Mutant Strains Mice Mutate Mutation Nematoda Nervous system structure Neuraxis Neurons Pattern Pharmacology Phenotype Phosphorylation Play Population Potassium Channel Probability Proteins RNA Splicing Reflex action Research Rodent Rodent Model Role Rotarod Performance Test Sedation procedure Severities Shapes Testing Texas Time Voltage-Gated Potassium Channel Water Withdrawal alcohol behavior alcohol effect alcohol response alcohol sensitivity alcohol testing austin behavior test behavioral impairment behavioral response drinking drinking behavior experimental study fly hypnotic in vivo indexing innovation large-conductance calcium-activated potassium channels mouse model novel patch clamp preference prevent protein function receptor response sedative side effect voltage

项目摘要

Project Summary Alcohol has wide-reaching effects on the nervous system. The mechanism of action for alcohol is complex, where alcohol interacts specifically and non-specifically with many targets (e.g. receptors, lipids, extra-cellular matrix, protein function, etc.). However, the fundamental mechanisms that underlie the effects of alcohol on different behaviors are poorly understood. Thus, more research is required to identify the key molecules essential for different alcohol behaviors (sensitivity to sedation, withdrawal, tolerance, and drinking) that may be targeted to yield new treatments. We focus here on the role of the BK channel, a calcium and voltage-gated potassium channel, in behavioral responses to alcohol. The highly conserved BK potassium channel is a direct target of ethanol that might be modified to reduce alcohol behaviors with minimal side effects. Unbiased genetic screens revealed that the BK channel represented by far the most important ethanol target for intoxication in Caenorhabditis elegans. Acute ethanol directly activates the BK channel to depress general neuronal activity and behaviors in worm. The BK channel was subsequently implicated as important in various behavioral responses to alcohol in flies, rodents and humans. New research also suggests that the channel may be targeted in a way to minimize side effects. A novel BK channel mutation has been identified (T352I) that prevents effects of intoxication and alcohol withdrawal in a C. elegans model. This mutation alters a single residue that is conserved in worm, mouse and human BK channels. Patch-clamp recordings confirmed that the human BK T352I channel was insensitive to activation by ethanol, but otherwise had normal conductance, K+ selectivity, and only subtle differences in voltage dependence. The T352I mutation may alter a binding site for ethanol and/or interfere with ethanol-induced conformational changes critical for behavioral responses. These results suggest that knocking in the T352I mutation in rodent models may alter ethanol-dependent behaviors without causing gross behavioral impairments, which would advance our understanding of the role of the BK channel in different ethanol-mediated behaviors. For this proposal, we will determine whether the BK channel represents a major target of ethanol to modify behaviors in mammals. This will be done by performing quantitative analysis of alcohol-related behaviors in our new mouse engineered with the BK T352I mutation via CRISPR/Cas9; this mouse was generated with our collaborators Drs. John Pierce, Gregg Homanics, and William Shawlot. We will test whether the T352I mutation reduces sensitivity to ethanol sedation, withdrawal, tolerance, and drinking. We hypothesize that the reduced sensitivity seen in C. elegans carrying the T352I BK mutation will be recapitulated in more complex but analogous behaviors in mutant mice. These studies have the potential to determine whether the BK channel represents a major contributor across different alcohol behaviors in mammals. They will also help elucidate the molecular role of the BK channel in alcohol withdrawal and its potential as a treatment avenue during withdrawal.

项目概要酒精对神经系统有广泛的影响。酒精的作用机制很复杂酒精与许多靶标（例如受体、脂质、细胞外物质）发生特异性和非特异性相互作用。基质、蛋白质功能等）。然而，酒精影响的基本机制人们对不同的行为知之甚少。因此，需要更多的研究来确定关键分子对于不同的酒精行为（对镇静、戒断、耐受和饮酒的敏感性）至关重要，这些行为可能有针对性地产生新的治疗方法。我们在这里重点关注 BK 通道的作用，它是一种钙和电压门控通道钾通道，在对酒精的行为反应中。高度保守的 BK 钾通道是直接乙醇的目标可能会被修改以减少饮酒行为，同时副作用最小。不偏不倚遗传筛选显示，BK 通道是迄今为止最重要的乙醇靶点秀丽隐杆线虫中毒。急性乙醇直接激活BK通道抑制全身蠕虫的神经元活动和行为。 BK 频道随后在各种事件中被认为很重要苍蝇、啮齿动物和人类对酒精的行为反应。新的研究还表明，该渠道可以以尽量减少副作用的方式进行针对性治疗。已鉴定出一种新的 BK 通道突变 (T352I) 可以防止线虫模型中中毒和酒精戒断的影响。这种突变改变了一个单一的蠕虫、小鼠和人类 BK 通道中保守的残留物。膜片钳记录证实人 BK T352I 通道对乙醇激活不敏感，但电导正常，K+ 选择性，并且电压依赖性上只有细微的差别。 T352I 突变可能会改变结合位点乙醇和/或干扰乙醇诱导的对于行为反应至关重要的构象变化。这些结果表明，敲除啮齿动物模型中的 T352I 突变可能会改变乙醇依赖行为不会造成严重的行为障碍，这将增进我们对 BK 作用的理解不同乙醇介导的行为中的通道。对于这个提案，我们将确定BK通道是否代表乙醇改变哺乳动物行为的主要目标。这将通过执行来完成对我们的带有 BK T352I 突变的新小鼠的酒精相关行为进行定量分析 CRISPR/Cas9；该鼠标是与我们的合作者 Drs. 一起制作的。约翰·皮尔斯、格雷格·霍曼尼克斯和威廉·肖洛特.我们将测试 T352I 突变是否会降低对乙醇镇静、戒断、耐受、饮酒。我们假设携带 T352I BK 的线虫中的敏感性降低突变将在突变小鼠的更复杂但类似的行为中得到体现。这些研究有确定 BK 通道是否代表不同酒精的主要贡献者的潜力哺乳动物的行为。它们还将有助于阐明 BK 通道在酒精戒断中的分子作用及其作为戒断期间治疗途径的潜力。