The molecular basis for the action of ethanol on the BK channel

描述(由申请人提供)：以前的研究已经证明酒精滥用的发展中有很强的遗传成分。具体地说，在第一次饮酒时对醉酒抵抗力高的人处于危险之中。这表明酒精或其下游调节行为醉酒的效应物的蛋白质靶标对个人滥用酒精的可能性至关重要。一种高度保守的靶蛋白是大电导钾(BK)通道，它是行为中毒和耐受的关键媒介。在包括线虫、啮齿动物和人类在内的许多物种中，BK通道都被低水平的酒精(~20 mM)激活，这相当于法定的中毒水平。在小鼠中，这一通道有助于行为陶醉和耐受性。BK通道的功能突变导致人类对酒精过敏。此外，在模式生物秀丽线虫中进行的基因筛查发现，BK通道的蠕虫直系同源基因SLO-1的零突变产生了对行为中毒的极端水平的抵抗。为了在分子水平上阐明乙醇与BK通道的相互作用 在这个水平上，我们正在使用两种遗传方法对线虫进行研究，以发现蠕虫和人类BK通道基因中新的非零突变，这些突变会导致对行为中毒的抵抗。我们将能够在蠕虫中研究人类BK通道，因为我们已经通过拯救具有人类BK通道的SLO-1(空)线虫中的乙醇敏感性而使蠕虫“人性化”。首先，我们将进行专门的基因筛查，以分离BK通道蠕虫版本中导致对醉酒行为抵抗的新的非零突变。其次，我们将对人BK通道基因进行定向随机突变，并将突变的基因转化到SLO-1(空)线虫中。转化后的线虫将被测试对醉酒的行为抵抗力。对于这两种方法，非零候选突变将识别对行为醉酒至关重要的基因的关键部分(S)。我们将区分NULL和 通过分析运动姿势和随后的DNA测序发现非零突变。到目前为止，从第一种方法获得的22个突变体中，有一个已被鉴定为候选非零突变体。在鉴定出非零突变体后，我们将在体内进行膜片钳记录，以评估该突变如何在单通道活动水平上改变基本的BK通道功能和对酒精的反应。这项研究将提供关于乙醇调节BK通道的关键残基的知识，从而导致跨物种的行为中毒，并更好地理解乙醇如何在分子水平上与离子通道相互作用。 与公共健康相关：大电导钾(BK)通道对许多物种的行为中毒和/或耐受性至关重要。这项研究的目的是调查BK通道上的哪些残基在分子水平上对乙醇的调节以及行为中毒起关键作用。这项研究的结果将有助于在分子水平上更好地了解乙醇与离子通道的相互作用，并对治疗酒精滥用有一定的意义。

DESCRIPTION (provided by applicant): Previous research has demonstrated a strong genetic component in the development of alcohol abuse. Specifically, people with a high resistance to intoxication at the time of their first drink are at risk. This suggests protein targets of alcoholor their downstream effectors that mediate behavioral intoxication are critical for the potential of a individual to abuse alcohol. A highly conserved target protein that is emerging as a key mediator for behavioral intoxication and tolerance is the big conductance potassium (BK) channel. The BK channel is activated by low levels of alcohol (~20 mM) across many species including C. elegans, rodent and human, which is equivalent to the legal level of intoxication. In mice, this channel contributes to both behavioral intoxication and tolerance. A gain-of function mutation in the BK channel results in hypersensitivity to alcohol in humans. In addition, a genetic screen performed in the model organism C. elegans discovered that null mutations in the worm ortholog of the BK channel, SLO-1, produced an extreme level of resistance to behavioral intoxication. In order to elucidate the interaction of ethanol and the BK channel at the molecular level, we are using two genetic approaches with C. elegans to uncover novel non-null mutations in the worm and human BK channel gene that result in resistance to behavioral intoxication. We will be able to study the human BK channel in the worm because we have "humanized" the worm by rescuing ethanol sensitivity in a slo-1(null) C. elegans with the human BK channel. First, we will perform a specialized genetic screen to isolate novel non-null mutations in the worm version of BK channel that result in behavioral resistance to intoxication. Second, we will carry out targeted random mutagenesis on the human BK channel gene, and transform the mutated gene into slo-1(null) C. elegans. Transformed C. elegans will be tested for behavioral resistance to intoxication. For both approaches, non-null candidate mutations will identify key portion(s) of the gene that are critical for behavioral intoxication. We will distinguish null from non-null mutations by analysis of locomotory posture and subsequent DNA sequencing. So far, one of the 22 mutants obtained from the first approach has been identified as a candidate non-null mutant. After non-null mutants are identified, we will perform in vivo patch-clamp recordings to assess how the mutation alters basal BK channel function and response to alcohol at the level of single-channel activity. This study will provide knowledge on what residues are critical for ethanol modulation of the BK channel that results in behavioral intoxication across species, and a better understanding of how ethanol interacts with ion channels at the molecular level. PUBLIC HEALTH RELEVANCE: The big conductance potassium (BK) channel is critical for behavioral intoxication and/or tolerance across many species. The goal of this study is to investigate what residues on the BK channel are critical for ethanol modulation at the molecular level, and behavioral intoxication. Results from this study will lead to a better understanding of how ethanol interacts with ion channels at the molecular level, and have implications for treating alcohol abuse.