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

乙醇对 BK 通道作用的分子基础

• 批准号: 8398535
• 负责人: Scott J Davis
• 金额: $ 3.81万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8398535
• 关键词: Alcohol abuse; Alcohols; Alleles; Amino Acids; Animal Model; Behavioral; Binding; Caenorhabditis elegans; Code; DNA Sequence; Development; Ethanol; Generations; Genes; Genetic; Genetic Screening; Genome; Goals; Human; Hypersensitivity; Individual; Intoxication; Ion Channel; Knowledge; Lead; Legal; Libraries; Locomotion; Mediating; Mediator of activation protein; Methods; Molecular; Mus; Mutagenesis; Mutate; Mutation; Mutation Analysis; Neurons; Nucleotides; Oncogenes; Orthologous Gene; Physiological; Posture; Potassium; Proteins; Publishing; Research; Resistance; Rodent; Testing; Time; Transgenic Organisms; alcohol abuse therapy; alcohol effect; alcohol measurement; alcohol response; alcohol sensitivity; base; behavior test; design; egg; gain of function mutation; high risk drinking; in vivo; insight; large-conductance calcium-activated potassium channels; mutant; novel; null mutation; patch clamp; promoter; response

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.

描述（由申请人提供）：先前的研究已经证明，酒精滥用的发展具有很强的遗传因素。具体来说，第一次饮酒时对中毒具有较高抵抗力的人面临风险。这表明酒精的蛋白质靶标或其介导行为中毒的下游效应器对于个体滥用酒精的可能性至关重要。大电导钾 (BK) 通道是一种高度保守的靶蛋白，它正在成为行为中毒和耐受性的关键介质。许多物种（包括线虫、啮齿动物和人类）的 BK 通道会被低浓度的酒精 (~20 mM) 激活，这相当于法定的中毒浓度。在小鼠中，该通道有助于行为中毒和耐受。 BK 通道的功能获得性突变会导致人类对酒精过敏。此外，在模型生物秀丽隐杆线虫中进行的基因筛选发现，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高水平的抵抗力。 为了从分子水平上阐明乙醇与BK通道的相互作用 在水平上，我们正在对秀丽隐杆线虫使用两种遗传方法来发现线虫和人类 BK 通道基因中新的非无效突变，这些突变可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，因为我们通过使用人类 BK 通道拯救 slo-1（无效）线虫中的乙醇敏感性，使该蠕虫“人性化”。首先，我们将进行专门的基因筛选，以分离蠕虫 BK 通道中新的非无效突变，这些突变会导致对中毒的行为抵抗。其次，我们将对人类BK通道基因进行定向随机突变，将突变基因转化到slo-1(null)秀丽隐杆线虫中。将测试转化的秀丽隐杆线虫对中毒的行为抵抗力。对于这两种方法，非无效候选突变将识别对行为中毒至关重要的基因关键部分。我们将 null 与 通过运动姿势分析和随后的 DNA 测序来确定非无效突变。到目前为止，第一种方法获得的 22 个突变体中的一个已被确定为候选非零突变体。鉴定出非无效突变体后，我们将进行体内膜片钳记录，以评估突变如何改变基础 BK 通道功能以及单通道活性水平对酒精的反应。这项研究将提供关于哪些残留物对于 BK 通道的乙醇调节至关重要的知识，从而导致跨物种的行为中毒，并更好地了解乙醇如何在分子水平上与离子通道相互作用。 公共健康相关性：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。本研究的目的是调查 BK 通道上的哪些残基对于分子水平上的乙醇调节和行为中毒至关重要。这项研究的结果将有助于更好地了解乙醇如何在分子水平上与离子通道相互作用，并对治疗酒精滥用具有影响。