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了酒精滥用的发展中有很强的遗传成分。具体来说，在第一次饮酒时对醉酒具有高抵抗力的人处于危险之中。这表明，酒精的蛋白质靶点或其下游效应物介导的行为中毒是一个人滥用酒精的潜力至关重要。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介质。BK通道在包括C.线虫、啮齿动物和人类，这相当于法律的中毒水平。在小鼠中，该通道有助于行为中毒和耐受。BK通道中的功能获得性突变导致人类对酒精过敏。此外，在模式生物C. elegans发现BK通道的蠕虫直系同源物SLO-1中的无效突变产生了对行为中毒的极端抵抗力。 为了阐明乙醇与BK通道在分子水平上的相互作用， 水平，我们使用两种遗传方法与C。elegans发现蠕虫和人类BK通道基因中的新的非无效突变，导致对行为中毒的抵抗。我们将能够研究蠕虫中的人类BK通道，因为我们通过拯救α-1（空）C中的乙醇敏感性来"人源化"蠕虫。elegans与人类BK通道。首先，我们将进行一个专门的遗传筛选，以分离在BK通道的蠕虫版本中的新的非无效突变，导致对中毒的行为抗性。第二，我们将对人BK通道基因进行定向随机突变，将突变基因转化入人BK-1（null）C。优雅变形C将测试秀丽线虫对中毒的行为抵抗力。对于这两种方法，非空候选突变将识别对行为中毒至关重要的基因的关键部分。我们将null与 通过分析运动姿势和随后的DNA测序确定非无效突变。到目前为止，从第一种方法获得的22个突变体中的一个已被鉴定为候选非无效突变体。在非无效突变体被确定后，我们将进行体内膜片钳记录，以评估突变如何改变基础BK通道功能和单通道活性水平对酒精的反应。这项研究将提供什么样的残基是关键的BK通道，导致跨物种的行为中毒乙醇调制的知识，以及更好地了解乙醇如何在分子水平上与离子通道相互作用。 公共卫生相关性：大电导钾（BK）通道对于许多物种的行为中毒和/或耐受性至关重要。本研究的目的是研究BK通道上的哪些残基在分子水平上对乙醇调节和行为中毒至关重要。这项研究的结果将导致更好地了解乙醇如何在分子水平上与离子通道相互作用，并对治疗酒精滥用有影响。

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.