PROJECT SUMMARY Persistent pain affects millions of people in America and costs billions of dollars in healthcare and lost productivity annually. The most concerning aspect of pain management is the widespread use of opioids which has led to an addiction epidemic and the deaths of tens of thousands of Americans each year. These problems necessitate the development of novel therapeutics in the treatment of chronic pain, and a growing body of literature suggests that transient receptor potential ankyrin 1 (TRPA1) is a bona fide drug target for reducing chronic pain. TRPA1 is expressed in sensory neurons where it generates acute pain signals in response to noxious, electrophilic compounds. However, TRPA1’s promise as a drug target comes from its role as a positive regulator in a pain and inflammation loop where dysregulation of this feedback loop could underlie the transition from acute to chronic pain. Animal studies utilizing knockout mice show that TRPA1 activity is necessary for mechanical and thermal hypersensitivity produced from tissue injury as well as asthma-induced airway inflammation supporting this model. The overarching goal of this proposal is to better understand TRPA1 regulation with special focus being given to the mechanisms of calcium regulation. This has been a contentious topic in the field with some groups providing evidence that TRPA1 activity is regulated by binding calcium ions directly with no contribution from calmodulin (CaM) while others have shown CaM binds to TRPA1 and regulates its activity. My preliminary results suggest CaM binds to TRPA1 and is modulated by calcium ion binding as well as flexible cytoplasmic domains of TRPA1. I hypothesize there are multiple TRPA1 domains involved in CaM binding that CaM differentially engages at distinct calcium concentrations. I will test this hypothesis using mutant TRPA1 constructs that have impaired ability to bind calcium at the S2-S3 loop or have truncations of the flexible cytoplasmic regions at the N and C-termini with a combination biochemical, biophysical, and structural biology techniques. Experiments proposed in Aim 1 will determine the overall effect of the S2-S3 calcium binding loop and CaM on TRPA1 in two parts. The first set of experiments will determine the binding affinity of CaM to TRPA1 and the S2-S3 triple mutant at varying calcium concentrations. The second set of experiments will determine the binding stoichiometry of CaM to TRPA1 at varying calcium concentrations. Aim 2 seeks to identify the flexible cytoplasmic regions of the N and C-termini involved in CaM binding. The structural changes that CaM imparts on TRPA1 and how these different regions modulate CaM binding will be explored in Aim 3. Overall, I will perform a comprehensive study investigating the relationship between CaM binding, the S2- S3 calcium binding site, and the flexible cytoplasmic regions. A more complete picture of TRPA1 regulation will provide new opportunities for rational drug desig...