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.
This project will identify novel peptide modulators of the BK channel, a relevant target for the treatment of alcohol abuse. In the future, these bioactive peptides will help to fine-tune our understanding of how the BK channel contributes to ethanol's actions, and will aid in the development of pharmaceuticals to treat people who abuse alcohol. )
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|Chen, Xixi; Yan, Jiusheng; Aldrich, Richard W (2014) BK channel opening involves side-chain reorientation of multiple deep-pore residues. Proc Natl Acad Sci U S A 111:E79-88|
|Topper, Stephen M; Aguilar, Sara C; Topper, Viktoria Y et al. (2014) Alcohol disinhibition of behaviors in C. elegans. PLoS One 9:e92965|
|Davis, Scott J; Scott, Luisa L; Hu, Kevin et al. (2014) Conserved single residue in the BK potassium channel required for activation by alcohol and intoxication in C. elegans. J Neurosci 34:9562-73|