The Role of BK Channel Composition in Alcohol Tolerance and Consumption

描述(由申请人提供)：行为耐受性是接触大多数滥用药物的标志。它的特点是药物效果随着时间的推移而减少，要么是通过改变药物的新陈代谢，要么是在功能上，即药物的效果在不改变浓度的情况下减少。事实上，人类急性行为耐受性增强的存在可以作为未来发展为酒精中毒的可能性的标志。大电导钾离子通道(BK)越来越被认为是酒精作用的重要靶点，无论是在药物的醉酒作用中，还是在对药物的适应(耐受)方面。这一建议是基于令人兴奋的新数据，该数据确认BK通道Beta4亚基是酒精耐受和消费的重要中介，并提供了一个动物模型来研究分子和行为耐受与饮酒之间的关系。我们将利用基因工程小鼠，在这些小鼠中，Beta4亚单位要么存在，要么“被敲除”。在目标1中，我们验证了BK beta4通过一种涉及激酶和相关信号通路的机制影响急性耐受的假设。电生理学实验将探索在BK急性耐受中，在表达系统和纹状体神经元中的激酶的作用，这在药物奖赏和成瘾中是重要的。在目标2中，我们检验了假设(基于初步数据)，即β4‘S对快速耐受的影响不会像急性耐受那样明显。这些研究将利用电生理学、分子生物学和成像技术，并将在分子、细胞和行为层面进行探索。目标2将包括对Beta4在这一发现中的作用的研究，在我们的初步数据中描述，在纹状体神经元中，有一个在酒精暴露3到6小时后被激活的“开关”，它从根本上改变了酒精戒断后的耐受持续时间。这对酒精依赖的形成是饮酒习惯的一种影响。最后，在目标3中，我们检验了假设，即S对酒精消费的影响将反映对酒精偏好和奖励等行为参数的影响，这两个因素都会影响消费。“在黑暗中饮用”(DID)范式的两瓶选择变体将被用来测量Beta4KO和WT小鼠对乙醇和水的偏好。此外，我们将使用条件性位置偏爱(CPP)试验来测量乙醇在WT和Beta4KO小鼠中的奖励特性。总之，这些实验将有助于确定Beta4亚单位和急性耐受性在酒精消费背后的动机行为中的作用。 公共卫生相关性：酗酒是公共卫生领域最重要的问题之一。耐受性是随着酒精的使用而发展起来的，它可能与药物依赖和成瘾的发展有关。在这项提议中，我们追求令人兴奋的新数据，这些数据表明BK通道Beta4亚单位蛋白在控制酒精耐受性和饮酒方面发挥了作用。

DESCRIPTION (provided by applicant): Behavioral tolerance is a hallmark of exposure to most drugs of abuse. It is characterized by reduced drug effects over time, either via altered metabolism of the drug, or functionally, whereby the effects of the drug decrease in spite of unaltered concentration. Indeed, the presence of enhanced acute behavioral tolerance in humans can serve as a marker for the likelihood of a future development of alcoholism. The large conductance potassium ion channel (BK) has become increasingly recognized as an important target of alcohol action, both in the intoxicating actions of the drug, as well as in adaptation to the drug (tolerance). This proposal is based upon exciting new data which identifies the BK channel beta4 subunit as an important mediator of alcohol tolerance and consumption, and provides an animal model to examine the relationship between molecular and behavioral tolerance, and consumption. We will make use of genetically engineered mice, in which the beta4 subunit is either present or "knocked out". In Aim 1, we test the hypothesis that BK beta4 influences acute tolerance via a mechanism involving kinases and associated signaling pathways. Electrophysiological experiments will probe the role of kinases in BK acute tolerance in expression systems and in neurons of the striatum, which is important in drug reward and addiction. In Aim 2, we test the hypothesis (based upon preliminary data) that beta4's influence on rapid tolerance will not be as pronounced as in acute tolerance. These studies will utilize electrophysiological, molecular biology, and imaging techniques, and will probe at the molecular, cellular, and behavioral levels. Aim 2 will include studies on the role of beta4 in the finding, described in our preliminary data that in striatal neurons, there is a "switch" which is activated after 3 to 6 hrs of alcohol exposure, which radically changes the duration of tolerance after alcohol withdrawal. This has implications for the development of alcohol dependency as a function of drinking habits. Finally, in Aim 3, we test the hypothesis that beta4's influence on alcohol consumption will reflect influences on behavioral parameters such as alcohol preference and reward, both known to influence consumption. A two bottle choice variant of the "drinking in the dark" (DID) paradigm will be utilized to measure ethanol vs. water preference in beta4 KO and WT mice. In addition, we will measure the rewarding properties of ethanol in WT and beta4 KO mice using a conditioned place preference (CPP) assay. Together, these experiments will help identify the role of the beta4 subunit and acute tolerance in motivational behaviors underlying alcohol consumption. PUBLIC HEALTH RELEVANCE: Alcoholism represents one of the most important problems in the public health arena. Tolerance, which develops with alcohol use, may be related to the development of drug dependency and addiction. In this proposal, we pursue exciting new data which indicates a role for the BK channel beta4 subunit protein in the control of alcohol tolerance and consumption.