Adaptations to chronic activation of BK channels by ethanol: Contribution to dependence and tolerance

乙醇对 BK 通道慢性激活的适应：对依赖性和耐受性的贡献

• 批准号: 9895344
• 负责人: Candice Contet
• 金额: $ 24.76万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-15 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9895344
• 关键词: Acute; Affect; Affective; Alcohol consumption; Alcohol dependence; Alcoholic Intoxication; Alcohols; Alternative Splicing; Amygdaloid structure; Animal Model; Anti-Anxiety Agents; Automobile Driving; Brain; Brain region; Calcium; Calcium Signaling; Chronic; Collaborations; Data; Dependence; Development; Down-Regulation; Ethanol; Ethanol dependence; Exposure to; Habenula; Human; In Vitro; Inhalation; Investigation; Knock-in Mouse; Knock-out; Knockout Mice; Mass Spectrum Analysis; Mediating; Modeling; Molecular; Molecular Target; Motivation; Mus; Negative Reinforcements; Nuclear; Pathway Analysis; Phase; Population; Post-Translational Protein Processing; Potassium Channel; Prefrontal Cortex; Proteins; Proteome; Proteomics; RNA Interference; Research Project Grants; Risk Factors; Role; Structure; Testing; Time; Up-Regulation; Variant; Ventral Tegmental Area; Viral; Wild Type Mouse; Work; alcohol effect; alcohol sensitivity; alcohol use disorder; base; design; drinking; experimental study; heuristics; in vivo; individual variation; knock-down; large-conductance calcium-activated potassium channels; mouse model; mutant; novel; protein expression; response; vapor; voltage

SUMMARY Large conductance, voltage- and calcium-activated (BK) channels are a molecular target of ethanol. Our data indicate that ethanol-induced activation of BK channels facilitates the escalation of voluntary ethanol consumption in mice made dependent to ethanol. We therefore hypothesize that molecular adaptations resulting from chronic activation of BK channels by ethanol facilitate the progression to dependence, possibly by lowering ethanol sensitivity. Accordingly, our project aims to elucidate the molecular identity of BK-dependent adaptations (Aim 1, R21 phase) and to test their functional implication in the transition to dependence (Aim 2, R33 phase) and in the control of ethanol sensitivity (Aim 3, R33 phase). We will take advantage of a knockin mouse expressing BK channels that are insensitive to ethanol but function normally otherwise to identify molecular adaptations to chronic ethanol that selectively result from the action of ethanol on BK channels. Molecular adaptations that emerge in brain regions relevant to the motivational and affective effects of ethanol (ventral tegmental area, amygdala, prelimbic prefrontal cortex, and habenula) will be examined in a well-validated mouse model of ethanol dependence. We will leverage the unprecedented sensitivity and accuracy of data-independent acquisition mass spectrometry to quantify changes in protein abundance across the entire proteome. Furthermore, we will implement weighted correlation network analysis to identify proteins that are the most likely to drive concerted changes in abundance across modules of co-expressed proteins. Nine of these proteins will be selected at the end of the R21 phase for functional analysis during the R33 phase. We will use virally mediated RNA interference to knock down candidate proteins in targeted brain regions and evaluate the influence of these proteins on the time-course, amplitude and persistence of drinking escalation in ethanol-dependent mice. We predict that some of the proteins controlling drinking escalation will also control acute sensitivity to ethanol, such that their up- or down-regulation in ethanol-dependent mice would progressively decrease sensitivity to ethanol. Accordingly, we will also examine the impact of local protein knockdown on the reinforcing and anxiolytic effects of ethanol. Altogether, the proposed experiments are designed to identify novel molecular determinants of vulnerability to alcohol use disorders. Our proposal is relevant to the focus of FOA PAR-18-659 because the proteins identified in this project may pinpoint molecular mechanisms underlying differential sensitivity to alcohol in the human population.

概括 大电导，电压和钙激活（BK）通道是乙醇的分子靶标。我们的数据 表明乙醇诱导的BK通道的激活有助于自愿乙醇的升级 小鼠的消耗取决于乙醇。因此，我们假设导致的分子适应 乙醇从慢性激活BK通道促进依赖性，可能通过降低 乙醇敏感性。因此，我们的项目旨在阐明BK依赖性适应的分子身份 （AIM 1，R21阶段）并测试其在依赖性过渡中的功能含义（AIM 2，R33阶段） 并控制乙醇敏感性（AIM 3，R33期）。我们将利用敲击鼠标 表达对乙醇不敏感但通常功能以识别分子的BK通道 对慢性乙醇的适应性，从乙醇对BK通道的作用有选择地产生。分子 与乙醇的动机和情感作用相关的大脑区域中出现的适应 将在验证良好的小鼠中检查颗粒区域，杏仁核，前额叶皮层和Habenula） 乙醇依赖的模型。我们将利用独立于数据的前所未有的灵敏度和准确性 采集质谱法以量化整个蛋白质组蛋白质丰度的变化。 此外，我们将实施加权的相关网络分析，以识别最有可能的蛋白质 驱动共同表达蛋白质模块之间丰度的一致变化。这些蛋白质中的九种将 在R21阶段结束时选择在R33阶段进行功能分析。我们将使用病毒调解 RNA干扰以在靶向大脑区域击倒候选蛋白并评估这些蛋白质的影响 蛋白质在乙醇依赖性小鼠中饮酒升级的时期，幅度和持久性。我们 预测某些控制饮酒升级的蛋白质还将控制对乙醇的急性敏感性，例如 它们在乙醇依赖性小鼠中的上调或下调将逐渐降低对乙醇的敏感性。 因此，我们还将检查局部蛋白质敲低对增强和抗焦虑作用的影响 乙醇。共同设计的实验旨在鉴定新的分子决定因素 酒精使用障碍的脆弱性。我们的建议与FOA Par-18-659的重点有关，因为 该项目中鉴定出的蛋白质可以确定对酒精差异敏感性的分子机制 在人口中。