Identifying Druggable Sites in the Pain-Sensing TRPA1 Ion Channel

Abstract

Transient receptor potential (TRP) channels, found in all eukaryotes, are a family of cation channels vital for critical physiological processes, such as mediating the sensation of unanticipated pain. Most TRP channel activities are modulated by a diverse set of exogenous and endogenous ligands, several of which are important for human life. For example, general anesthetics, which are widely used in surgical procedures, have undesired effects by activating TRPA1, which functions as a pain-sensing ion channel. Upon administration, several noxious general anesthetics stimulate peripheral sensory neurons, which transduce pain and inflammation elicited by these widely used drugs. Although evidence points to TRPA1 as the major player in this pain pathway, where propofol binds in the channel is unclear. Here I present the first photocrosslinking strategy to identify the propofol binding sites in TRPA1. I designed synthetically expedient routes to three crosslinkable analogues of propofol, two of which are also functionalized with an alkyne affinity handle for bioorthogonal chemistry. Using mouse TRPA1 heterologously expressed in insect cells and fluorescence-based functional assays to compare propofol and the synthesized analogues, I determined that ortho-substitution, relative to the hydroxyl group, still allows for TRPA1 activation. Using the same activity assay, I conducted small molecule structure-function studies, introducing substituents replacing the phenolic hydroxyl group of propofol as well as adding or replacing substituents at the ortho and para positions. My results demonstrate the importance of hydrogen bonding in propofol activation of TRPA1 and provide evidence that halogen substitution can affect channel function. Photoaffinity labeling with a well-characterized propofol analogue, AziPm, which demonstrates the same dose-dependent and bimodal TRPA1 activation profile as propofol, identified two crosslinked residues in TRPA1. Both residues lie on the S6 helix, which, together with S5, forms the channel pore and gate of TRPA1. V954 faces a pocket previously identified as the A-9607079 antagonist binding site, while E969 lies near the intracellular mouth of the pore at the junction of S6 and the “TRP-domain” -helix, which is important for channel gating. These results provide the first direct identification of general anesthetic binding sites leading to TRPA1 activation.