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

描述（由申请人提供）：对多种物种的研究表明，大电导钙活化钾（BK）通道是乙醇的靶点，也是治疗酒精滥用的相关药理学靶点。开发特定的BK通道调节剂可用于描述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，我们将确定以乙醇依赖或独立方式改变BK通道活性的小肽。结合三位pi的专业知识，John Mihic， Jonathan Pierce-Shimomura和Richard Aldrich，我们开发了一种经过验证的方法来合理发现具有药理活性的肽。通过一系列的技术，该方法提供了一个从头到尾的搜索和表征新的BK信道调制器。我们将首先利用噬菌体展示技术鉴定与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，可以筛选数千万种肽，以确定它们与特定靶标的特异性结合能力。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点结合的肽。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类BK通道活性的能力。在存在和不存在乙醇的情况下，BK通道功能与运动能力之间的关系都很明确，这使得秀丽隐杆线虫成为快速测试BK通道肽药理活性的理想系统。Pierce- Shimomura及其同事先前的研究表明，急性暴露于药理学相关水平的乙醇通过增强BK通道活性来降低秀丽隐杆线虫的运动。此外，我们最近已经能够复制秀丽隐杆线虫对乙醇的运动敏感性，表达人类BK通道而不是天然通道。利用这种表达人类BK通道的秀丽隐杆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，同时也可以检测BK通道依赖性活性。最后，我们将测试这些肽对异种系统中表达的BK通道门控的影响。这些电生理实验将利用理查德·奥尔德里奇多年来研究离子通道生物物理学的经验。我们将利用我们之前对变构门控的理解来理解肽和乙醇对BK通道的调节机制。我们广泛的专业知识，个人多年的经验和在同一部门的物理距离将促进我们的合作努力，以确定和表征在存在和/或不存在乙醇的情况下调节BK通道功能的小肽。这些新的BK通道调节剂可能促进药物治疗的发展，以帮助更多的酒精滥用患者。

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.