Role of BK Channel Across Alcohol Behaviors

项目摘要 酒精对神经系统有广泛的影响。酒精的作用机制很复杂， 其中酒精与许多靶（例如受体、脂质、细胞外 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。我们在这里集中在BK通道的作用，钙和电压门控 钾离子通道，在酒精的行为反应。高度保守的BK钾通道是一个直接的 乙醇的目标，可能会修改，以减少酒精的行为与最小的副作用。无偏 基因筛选显示，BK通道代表了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， 神经活动和行为。BK通道随后被认为在各种 在果蝇、啮齿动物和人类中对酒精的行为反应新的研究还表明， 可以以最小化副作用的方式进行靶向治疗。已经鉴定了一种新的BK通道突变（T352 I） 可以防止C型糖尿病患者的醉酒和戒酒。elegans模型这种突变改变了一个单一的 在蠕虫、小鼠和人BK通道中保守的残基。膜片钳记录证实， 人BK T352 I通道对乙醇激活不敏感，但具有正常电导，K+ 选择性，并且仅在电压依赖性方面存在细微差异。T352 I突变可能改变了T352 I的结合位点。 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，在啮齿动物模型中敲入T352 I突变可能会改变乙醇依赖性行为 而不会造成严重的行为障碍，这将促进我们对BK作用的理解。 不同乙醇介导的行为。对于这个建议，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 我们的新小鼠中与酒精相关的行为的定量分析， CRISPR/Cas9;这只小鼠是由我们的合作者John Pierce博士，Gregg Homanics博士和 威廉·肖洛特我们将测试T352 I突变是否会降低对乙醇镇静、戒断的敏感性， 宽容和饮酒。我们推测，在C。携带T352 I BK 突变将在突变小鼠中以更复杂但类似的行为重演。这些研究 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。他们还将有助于阐明BK通道在酒精戒断中的分子作用 以及其作为戒断治疗途径的潜力。

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.