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

摘要 大电导、电压和钙激活(BK)通道是乙醇的分子靶点。我们的数据 提示乙醇诱导的BK通道激活促进了自愿性乙醇的升级 使小鼠对乙醇产生依赖的消耗。因此，我们假设分子适应会导致 乙醇对BK通道的慢性激活促进了向依赖的进展，可能是通过降低 对酒精敏感。因此，我们的项目旨在阐明BK依赖适应的分子同一性 (目标1，R21阶段)，并测试它们在向依赖转变过程中的功能暗示(目标2，R33阶段) 乙醇敏感性的控制(目标3，R33期)。我们会利用敲门的老鼠 表达对乙醇不敏感但功能正常的BK通道以识别分子 对慢性乙醇的适应，选择性地由乙醇对BK通道的作用而产生。分子 与酒精的激励和情感效应(腹侧)相关的脑区出现的适应 被盖区、杏仁核、前额叶皮质和缰核)将在经过充分验证的小鼠身上进行检查。 酒精依赖模型。我们将利用独立于数据的前所未有的敏感度和准确性 获取质谱仪以量化整个蛋白质组中蛋白质丰度的变化。 此外，我们将实施加权相关网络分析来识别最有可能的蛋白质 以推动共表达蛋白质模块间丰度的协调变化。其中九种蛋白质会 在R21阶段结束时被选择用于R33阶段期间的功能分析。我们将使用病毒介体 RNA干扰在靶向脑区敲除候选蛋白质并评估其影响 蛋白质对酒精依赖小鼠饮酒升级的时程、幅度和持久性的影响。我们 预测一些控制饮酒升级的蛋白质也将控制对酒精的急性敏感性，例如 在酒精依赖的小鼠中，它们的上调或下调将逐渐降低对乙醇的敏感性。 因此，我们还将研究局部蛋白质敲除对增强和缓解焦虑效果的影响。 乙醇。总之，拟议的实验旨在确定新的分子决定因素 易受酒精使用障碍的影响。我们的建议与FOA PAR-18-659的重点相关，因为 该项目中确定的蛋白质可能精确定位酒精不同敏感性的分子机制。 在人类人口中。

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.