Proteomic Analysis of Protein Partners that Govern TRPA1 Trafficking and Functionality

Roughly one third of Americans suffer from chronic pain, yet little progress has been made to develop new pain remedies beyond non-steroidal anti-inflammatory agents and opioids. While opioids can be effective analgesic agents, these drugs harbor poor side effects and the risk of addiction because opioid receptors are expressed in neurons and brain regions outside the pain producing somatosensory neural network. An attractive therapeutic approach to develop new analgesic and anti-inflammatory agents, which will not harbor these poor side effects, is to identify receptors predominantly expressed in peripheral nociceptor neurons that initiate pain signals. One such receptor is the wasabi receptor, TRPA1, which is activated directly by a diverse panel of environmental and endogenous chemical irritants, as well as indirectly by pro-inflammatory mediators downstream of several G-protein coupled receptors, to originate painful sensations. Pain is not a single entity, but instead has many causes, types, and symptoms, including mechanical pain, temperature hypersensitivity, and pain from chronic inflammation or some chemotherapeutic agents. Despite these diversities, TRPA1 plays a central role in each etiology to generate pain signals. Indeed, two identified gain-of-function human TRPA1 natural variants, characterized by pronounced pain phenotypes in patients, provide strong evidence for the critical role of TRPA1 in human pain physiology. Accordingly, TRPA1 has been deemed a bona fide therapeutic target for analgesic and anti-inflammatory agent development, however, all known TRPA1 antagonists have failed in clinical trials due to poor pharmacokinetics. Despite its importance to human pain, little is known about how TRPA1 is regulated. A detailed understanding of the diverse mechanisms by which TRPA1 activity is modulated could uncover novel avenues for therapeutic agent development that may fare better in clinical trials.

Plasma membrane localization of TRPA1 is increased after direct channel activation or indirect activation by pro-inflammatory mediators, which could contribute to neuronal sensitization and, if unchecked, to the development of chronic pain. Nonetheless, little is known about how TRPA1 trafficking is regulated. Here, we propose to use BioID2-fused TRPA1 constructs in combination with proteomics analysis to identify interacting and proximity protein partners that govern proper channel trafficking and functionality, as our preliminary data suggests these processes are decoupled in TRPA1. In our first aim, we intend to identify protein partners necessary to mediate TRPA1 plasma membrane trafficking by using a nonfunctional TRPA1 natural variant when it is expressed alone or co-expressed with wild type protein. We recently discovered this nonfunctional natural variant exhibits aberrant trafficking alone and confers gain-of-function by assembling with wild type TRPA1 subunits to form hyperactive channels that are functional at the plasma membrane, though their trafficking route is unclear. In our second aim, we want to identify interacting and proximity protein partners necessary to confer channel functionality using wild type, functional yet N-linked glycosylation incompetent, or nonfunctional yet properly trafficking TRPA1 variants. The machinery involved in both of these processes are predicted to interact with the channel on its cytoplasmic face, which could uncover novel druggable sites that are distinct from the transmembrane regions targeted by current TRPA1 antagonists.