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

描述(申请人提供)：各种物种的研究表明，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靶标。开发特定的BK通道调节剂可以用来描述BK通道如何促进乙醇的作用，并用于开发治疗酒精滥用的药物。在这项提案中，我们将识别以乙醇依赖或独立的方式改变BK通道活性的小肽。结合三位PI约翰·米希克、乔纳森·皮尔斯-下村和理查德·奥尔德里奇的专业知识，我们开发了一种合理发现药理活性多肽的成熟方法。通过一系列技术，该方法提供了对新型BK通道调制器的从头到尾的搜索和表征。我们将首先利用噬菌体展示技术鉴定与BK通道结合的新型多肽。噬菌体展示技术是一种高效且经济的技术，可用于筛选数千万个多肽与特定靶点的结合能力。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标结合的多肽。使用该技术选择的多肽将进一步测试在乙醇存在或不存在的情况下影响在秀丽线虫中表达的人BK通道活性的能力。在乙醇存在和不存在的情况下，BK通道功能和运动能力之间的明确关系使线虫成为快速检测BK通道多肽药理活性的理想系统。Piells-Shimomura及其同事之前的工作表明，急性暴露于药理上相关水平的乙醇通过增强BK通道活性而减少线虫的运动。此外，我们最近已经能够在表达人BK通道而不是自然通道的线虫中复制对乙醇的运动敏感性。使用这种表达人BK通道的线虫作为肽功能的二级筛选，使我们能够利用噬菌体展示的高通量能力，同时还可以分析BK通道依赖的活性。最后，我们将测试这些肽对在异源系统中表达的BK通道的门控的影响。这些电生理实验将利用理查德·奥尔德里奇多年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多肽和乙醇对BK通道的调节机制。我们广博的专业知识、个人多年的经验以及在同一部门的实际接触将有助于我们共同努力，识别和表征在乙醇存在和/或不存在的情况下调节BK通道功能的小肽。这些新型的BK通道调节剂可能会促进药物治疗的发展，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.