A screen for peptides that alter BK channel-mediated alcohol intoxication

筛选改变 BK 通道介导的酒精中毒的肽

• 批准号: 8744352
• 负责人: Richard Aldrich
• 金额: $ 4.64万
• 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: 2012-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8744352
• 关键词: Acute; Affect; Alcohol abuse; Alcoholic Intoxication; Bacteriophages; Behavior; Behavioral; Binding; Biological Assay; Biological Availability; Biophysics; Blood; Blood Vessels; Brain; Caenorhabditis elegans; Calcium; Capsid Proteins; Cell Surface Proteins; Cells; Complement; Complex; Development; Drug Formulations; Enzymes; Ethanol; Ethanol dependence; Exposure to; Future; Human; Human Activities; In Vitro; Individual; Injectable; Intoxication; Ion Channel; Laboratories; Libraries; Locomotion; Mediating; Mediator of activation protein; Methodology; Methods; Nematoda; Neurotransmitter Receptor; Patients; Peptides; Phage Display; Pharmacologic Substance; Pharmacotherapy; Physiological; Potassium; Process; Randomized; Series; Site; System; Techniques; Testing; Transgenic Organisms; Work; alcohol abuse therapy; alcohol effect; alcohol exposure; alcohol response; alcohol sensitivity; base; cost effective; experience; genetic manipulation; high throughput screening; in vivo; large-conductance calcium-activated potassium channels; neuronal excitability; neurotransmission; novel; overexpression; research study; response; tool; voltage

DESCRIPTION (provided by applicant): Studies in a variety of species indicate that the large conductance calcium-activated potassium (BK) channel is a target of ethanol as well as a relevant pharmacological target for the treatment of alcohol abuse. The development of specific BK channel modulators can be used to delineate how the BK channel contributes to ethanol's actions and to develop pharmaceuticals for the treatment of alcohol abuse. In this proposal, we will identify small peptides that alter BK channel activity in an ethanol-dependent or independent fashion. Combining the expertise of three PIs, John Mihic, Jonathan Pierce-Shimomura and Richard Aldrich, we have developed a proven method for the rational discovery of pharmacologically active peptides. Through a series of techniques this methodology provides a start-to-finish search and characterization of novel BK channel modulators. We will first identify novel peptides that bind to BK channels using phage display. Phage display is an efficient and cost-effective technique to screen tens of millions of peptides for their abilities to bind to a particular target specifically. The Mihic laboratory has recently developed a new phage display technique to screen for peptides that bind to specific ion channel targets expressed in heterologous cells. Peptides selected using this technique will be further tested for the ability t affect the activity of human BK channels expressed in Caenorhabditis elegans in the presence or absence of ethanol. The well-defined relationship between BK channel function and locomotor ability both in the presence and absence of ethanol makes C. elegans an ideal system to rapidly test the pharmacological activity of peptides at the BK channel. Previous work by Pierce- Shimomura and colleagues showed that acute exposure to pharmacologically relevant levels of ethanol decreased locomotion in C. elegans by enhancing BK channel activity. Moreover, we have recently been able to replicate locomotor sensitivity to ethanol in C. elegans expressing the human BK channel instead of the native channel. Using this human BK channel-expressing C. elegans for a secondary screen of peptide function allows us to utilize the high-throughput power of phage display while also assaying for BK channel- dependent activity. Finally, we will test the effects of these peptides on the gating of BK channels expressed in a heterologous system. These electrophysiological experiments will capitalize on Richard Aldrich's many years of experience studying ion channel biophysics. We will use our previously developed understanding of allosteric gating to understand the mechanism of peptide and ethanol modulation of the BK channel. Our breadth of expertise, individual years of experience and physical proximity in the same department will facilitate our collaborative effort to identify and characterize small peptides that modulate BK channel function in the presence and/or absence of ethanol. These novel BK channel modulators may promote the development of pharmacotherapies to help a broader number of alcohol abusing patients.

描述（由申请人提供）：对多种物种的研究表明，大电导钙激活钾（BK）通道是乙醇的靶标，也是治疗酒精滥用的相关药理学靶标。特定 BK 通道调节剂的开发可用于描述 BK 通道如何促进乙醇的作用并开发用于治疗酒精滥用的药物。在本提案中，我们将鉴定以乙醇依赖性或独立方式改变 BK 通道活性的小肽。结合三位 PI（John Mihic、Jonathan Pierce-Shimomura 和 Richard Aldrich）的专业知识，我们开发了一种经过验证的方法来合理发现药理活性肽。通过一系列技术，该方法提供了新型 BK 通道调制器的从头到尾的搜索和表征。我们将首先使用噬菌体展示来鉴定与 BK 通道结合的新型肽。噬菌体展示是一种高效且经济的技术，可筛选数千万种肽，以确定它们与特定靶标特异性结合的能力。 Mihic 实验室最近开发了一种新的噬菌体展示技术，用于筛选与异源细胞中表达的特定离子通道靶标结合的肽。使用该技术选择的肽将进一步测试在存在或不存在乙醇的情况下影响秀丽隐杆线虫中表达的人BK通道活性的能力。在存在和不存在乙醇的情况下，BK 通道功能和运动能力之间明确的关系使秀丽隐杆线虫成为快速测试 BK 通道肽的药理活性的理想系统。 Pierce-Shimomura 及其同事之前的研究表明，急性暴露于药理学相关水平的乙醇会通过增强 BK 通道活性来降低线虫的运动能力。此外，我们最近能够在表达人类 BK 通道而不是天然通道的秀丽隐杆线虫中复制对乙醇的运动敏感性。使用这种表达人类 BK 通道的线虫进行肽功能的二次筛选，使我们能够利用噬菌体展示的高通量能力，同时还可以测定 BK 通道依赖性活性。最后，我们将测试这些肽对异源系统中表达的 BK 通道门控的影响。这些电生理学实验将利用理查德·奥尔德里奇多年研究离子通道生物物理学的经验。我们将利用之前对变构门控的理解来了解肽和乙醇调节 BK 通道的机制。我们广泛的专业知识、个人多年的经验以及在同一部门的物理接近将有助于我们的合作努力，以识别和表征在存在和/或不存在乙醇的情况下调节 BK 通道功能的小肽。这些新型 BK 通道调节剂可能会促进药物疗法的发展，以帮助更多的酗酒患者。