Role of BK Channel Across Alcohol Behaviors

项目摘要 酒精对神经系统有广泛的影响。酒精的作用机制是复杂的， 酒精与许多靶标(如受体、脂类、细胞外)发生特异性和非特异性相互作用 基质、蛋白质功能等)。然而，酒精对人体健康影响的基本机制 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 对不同的酒精行为(对镇静、戒断、耐受和饮酒的敏感性)是必不可少的，可能 以产生新的治疗方法为目标。这里我们重点研究BK通道的作用，BK通道是一种钙离子和电压门控通道 钾通道，在酒精的行为反应中。高度保守的BK钾通道是一个直接的 酒精的靶标，可以被修改以减少酒精行为，副作用最小。不偏不倚 基因筛查显示，BK通道是目前为止乙醇最重要的靶标 秀丽隐杆线虫中毒。急性乙醇直接激活BK通道抑制全身 蠕虫体内神经元的活动和行为。BK通道随后被认为在各种 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 可能以一种将副作用降至最低的方式进行靶向治疗。发现一种新的BK通道突变(T352I) 在线虫模型中，这可以防止醉酒和戒酒的影响。这种突变改变了单一的 在蠕虫、小鼠和人类BK通道中保守的残基。膜片钳记录证实 人BK-T352I通道对乙醇的激活不敏感，但具有正常的电导，K+ 选择性，仅在电压依赖性上有细微差别。T352I突变可能会改变结合位点 乙醇和/或干扰乙醇诱导的对行为反应至关重要的构象变化。这些 结果表明，敲击T352I突变可能会改变啮齿动物的酒精依赖行为 而不会造成严重的行为障碍，这将促进我们对BK的作用的理解 通道在不同的乙醇介导的行为中。对于这项提议，我们将确定BK频道是否 代表了乙醇改变哺乳动物行为的主要靶点。这将通过执行以下操作完成 我们的BK T352I突变新小鼠酒精相关行为的定量分析 CRISPR/Cas9；这只老鼠是与我们的合作者John Piels、Gregg Homanics和 威廉·肖洛特。我们将测试T352I突变是否会降低对酒精镇静、戒断、 宽容和饮酒。我们推测，携带T352I BK的线虫的敏感度降低 突变将在突变小鼠中更复杂但相似的行为中重现。这些研究已经 确定BK通道是否代表不同酒精的主要贡献者的可能性 哺乳动物的行为。他们还将有助于阐明BK通道在酒精戒断中的分子作用 以及它在戒断期间作为治疗途径的潜力。

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.