PROJECT SUMMARY The wasabi receptor, TRPA1, is a non-selective homotetrameric cation channel expressed in primary sensory neurons where its activation by noxious chemical irritants contributes to pain perception and local inflammation. Local inflammatory cues, in turn, sensitize sensory neurons to painful stimuli. Within the pain and local inflammation regulatory cycle, TRPA1 serves as a positive regulator and its dysregulation could contribute to the development of chronic pain. Genetic loss of TRPA1 in mice abrogates pain perception to chemical irritants, mechanical and thermal hypersensitivity produced from tissue injury, and asthma-induced airway inflammation supporting this model. Gain-of-function TRPA1 mutations cause congenital painful disorders in humans highlighting its direct role in pain perception. This makes understanding TRPA1 function and dysregulation highly significant. Basic science research in the Paulsen Laboratory broadly aims to determine molecular mechanisms of TRPA1 regulation and dysregulation by second messengers, local inflammatory cues, through protein-protein interactions, and as imparted by novel gain-of-function mutations. High-resolution TRPA1 structures exist in the open and closed states, which are sampled during normal channel activity. While they represent a major advance, these structures do not address the fundamental question of how TRPA1 becomes sensitized to confer channel hyperactivity in disease. Understanding these mechanisms would open the door to develop new targeted therapeutics. During the next 5 years, we will use complementary biochemical, biophysical, and structural biology approaches to determine the molecular basis of channel hyperactivity conferred by a novel gain-of-function TRPA1 mutation. Our preliminary data suggest this TRPA1 mutant protein co-assembles with wild type TRPA1 subunits to form hyperactive channels. We want to understand how the structural alterations introduced by this TRPA1 mutant affect channel function. Additionally, we will determine how TRPA1 is sensitized and/or activated by calcium and calmodulin. Many local inflammatory cues indirectly activate TRPA1 downstream of G-protein coupled receptors that promote intracellular calcium release. TRPA1 could bind calcium directly or the universal calcium sensor, calmodulin could mediate calcium sensing. Our preliminary data support an interplay of calcium and calmodulin in regulating TRPA1 and we want to understand how calmodulin binding works in concert with calcium-binding sites to confer TRPA1 calcium-dependent channel activity. Collectively, this work will enhance our understanding of regulation and dysregulation of TRPA1 at the molecular level and will uncover novel avenues for drug development to target aberrant channels in chronic pain and inflammatory conditions.