Elucidating the structural and molecular mechanisms of a TRPA1 channelopathic mutation

Yale University, New Haven CT

Investigators

Abstract

PROJECT SUMMARY/ABSTRACT Chronic pain affects roughly 100 million people per year in the United States and has led to increased healthcare costs and lost productivity. This has led to increased use of opioids and their misuse which has resulted in an opioid epidemic in individuals seeking to alleviate pain. Alternatives to opioids such as non-steroidal anti- inflammatory drugs possess adverse risks of their own if taken long-term. This highlights the pressing need to study pain at the source to aid in the development of therapeutics that provide safe, long-term relief for individuals with chronic pain without addiction risks. Transient receptor potential ankyrin 1 (TRPA1) is a homotetrameric, non-selective cation channel expressed in pain-sensing nociceptive neurons that responds to diverse chemical irritants. TRPA1 is at the forefront of pain perception, but its regulation remains largely unknown. Channelopathies, mutations in ion channels that lead to disease, are valuable tools that can aid in our understanding of TRPA1 regulation. A premature termination codon in TRPA1 (R919*), that truncates the final 201 amino acids, leads to the development of a severe hypersensitive pain disorder, (CRAMPT) syndrome. Our lab has found that co-expression of wildtype (WT) and R919* TRPA1 yields heteromeric channels with enhanced agonist sensitivity and currents. How incorporation of this mutant into heteromeric channels affects structure and function have yet to be defined. Furthermore, the abundance of each heteromer and which ones are physiologically relevant remains unknown. My overarching hypothesis is that all heteromeric WT-R919* TRPA1 channels form and ones with two or fewer R919* subunits are hyperactive. My project aims to resolve the structural and functional consequences of the R919* mutation on TRPA1 function, contributing to our understanding of how hyperactivation occurs. In Aim 1, I will purify heteromeric TRPV1 complexes to resolve high-resolution cryo-EM structures, in collaboration with the Mi lab, and for biochemical assays. Our lab has shown that an analogous mutation in TRP vanilloid 1 (TRPV1) shares a similar hyperactive profile. TRPV1 retains function upon purification while TRPA1 does not, presenting a tool to structurally understand the CRAMPT mutation. In Aim 2, I will use two-electrode voltage clamp (TEVC) to define the functional properties of heteromeric TRPA1 and TRPV1 channels. I will control WT:R919* TRPA1 cRNA ratios in Xenopus laevis oocytes to identify the relevant proportions required for hyperactivation. I will also test functionally isolated heteromeric TRPA1 and TRPV1 complexes by using TRPA1 constructs with reduced electrophilic activation and the purified TRPV1 sample from Aim 1. In Aim 3, I will determine the heteromeric TRPA1 complexes that form and their abundances. I will collaborate with the Bhattacharyya lab and utilize a single-molecule technique, nanoBleach, for imaging and stepwise photobleaching. Collectively, these studies seek to understand how the R919* mutation confers hyperactivity when in complex with WT TRPA1. Doing so will enhance our understanding of TRP channel conservation and reveal how perturbations in TRPA1 can be used to lead drug discovery for opioid alternatives.