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

基本信息

批准号：
8501154
负责人：
Richard Aldrich
金额：
$ 32.31万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
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/8501154
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 Signal Transduction 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 novel overexpression research study response tool voltage

项目摘要

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）：在各种物种中的研究表明，大型电导钙激活钾（BK）通道是乙醇的靶标，也是治疗酒精滥用的相关药理目标。可以使用特定BK通道调节剂的开发来描述BK通道如何对乙醇的作用做出贡献并开发用于治疗酒精滥用的药物。在此提案中，我们将确定以乙醇依赖或独立方式改变BK通道活动的小肽。结合了三个PI的专业知识，John Mihic，Jonathan Pierce-Shimomura和Richard Aldrich，我们开发了一种合理发现药理活性肽的验证方法。通过一系列技术，这种方法提供了新型BK通道调节剂的开始搜索和表征。我们将首先识别使用噬菌体显示与BK通道结合的新型肽。噬菌体显示是一种有效且具有成本效益的技术，可用于筛选数千万肽的能力，以与特定目标结合。 Mihic实验室最近开发了一种新的噬菌体显示技术，以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，以影响t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类BK通道的活性。在存在和不存在乙醇的情况下，BK通道功能与运动能力之间定义明确的关系使秀丽隐杆线虫成为迅速测试BK通道肽的药理活性的理想系统。 Pierce-Shimomura及其同事的先前工作表明，急性暴露于药理学相关水平的乙醇水平通过增强BK通道活性通过增强了秀丽隐杆线虫的运动。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通道的秀丽隐杆线虫进行肽功能的次级屏幕，使我们能够利用噬菌体显示的高通量功率，同时还要分析BK通道依赖性活动。最后，我们将测试这些肽对异源系统中表达的BK通道的门控的影响。这些电生理实验将利用理查德·奥尔德里奇（Richard Aldrich）研究离子通道生物物理学的多年经验。我们将利用先前对变构门控的理解来了解BK通道的肽和乙醇调节机制。我们在同一部门的专业知识，个体经验和身体接近的广度将有助于我们的协作努力，以识别和表征在乙醇存在和/或不存在乙醇的情况下调节BK渠道功能的小肽。这些新型的BK通道调节剂可能会促进药物疗法的发展，以帮助更广泛的滥用酗酒患者。