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

描述（由申请方提供）：在多种种属中进行的研究表明，大电导钙激活钾（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。在这个建议中，我们将确定小肽，改变BK通道活性的乙醇依赖性或独立的方式。结合三位PI，John Mihic，Jonathan Pierce-Shimomura和Richard Aldrich的专业知识，我们已经开发出一种经过验证的方法，用于合理发现生物活性肽。通过一系列的技术，这种方法提供了一个开始到结束的搜索和表征的新型BK通道调制器。我们将首先使用噬菌体展示鉴定与BK通道结合的新型肽。噬菌体展示是一种高效、经济的技术，可以筛选数千万肽段，以确定它们与特定靶点的特异性结合能力。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。将进一步测试使用该技术选择的肽在乙醇存在或不存在下影响秀丽隐杆线虫中表达的人BK通道活性的能力。BK通道功能和运动能力之间的明确关系，无论是在乙醇的存在和不存在，使C。elegans是一个理想的系统，以快速测试肽的药理活性在BK通道。Pierce-Shimomura及其同事先前的研究表明，急性暴露于与酒精浓度相关的乙醇会降低C。elegans通过增强BK通道活性。此外，我们最近已经能够复制运动敏感性乙醇在C。表达人类BK通道而不是天然通道的秀丽线虫。使用这种表达人类BK通道的C. elegans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，同时还测定BK通道依赖性活性。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。这些电生理学实验将利用理查德奥尔德里奇多年的经验，研究离子通道生物物理学。我们将使用我们以前开发的理解变构门控了解的BK通道的肽和乙醇调制的机制。我们在同一部门的专业知识，个人多年的经验和物理接近的广度将促进我们的合作努力，以确定和表征小肽调节BK通道功能的存在和/或不存在乙醇。这些新的BK通道调节剂可能会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.