Richard W. Aldrich - US grants

Project Summary As a central molecular hub in calcium signaling, calmodulin (CaM) is a key regulator of hundreds of target proteins including a wide range of ion channels. Essential for understanding the diversity of calmodulin mediated cellular processes is a thorough understanding of the physical relationships and interactions between calcium ions, the four EF- hand binding sites of calmodulin and the individual target proteins. We have developed new methods that allow the characterization of the four binding sites individually, including their binding affinities and cooperative interactions. Our previous work has shown, by site-specific binding measurements and evolutionary informatics that CaM's four EF-hand binding sites have different and distinct binding properties that have undergone strong selective pressures to remain different from each other. The overall goal of this proposal is to employ new state of the art experimental and analytic methods to understand the energetics and molecular mechanisms of calcium binding at each binding sites and how they are altered by occupancy at neighboring sites (cooperativity) and by binding to targets (transduction). We bring to these efforts powerful new experimental and theoretical approaches that we have developed that promise to lead to an unprecedented understanding of the CaM signal transduction mechanism that will have implications for ion channel regulation, calcium signaling and allosteric mechanisms in general. Our aims are to: (1) Determine by lanthanide luminescence spectroscopy the site-specific affinity and cooperativity of Ca2+ and Ln3+ binding to each of the four EF hands of free CaM in solution; (2) Determine how each of the four EF-hand ligand binding affinities are changed upon binding to specific target proteins or appropriate peptide fragments and how calmodulin peptide affinity and stoichiometry are modulated by calcium binding; (3) Determine the amino attributes that determine the unique binding properties of the four EF hand Ca2+ binding sites in CaM using lanthanide luminescence spectroscopy on calmodulin binding site chimeras.

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.