A novel TRPA1 gain-of-function variant associated with painful sensory neuropathy acts through a PIP2 gating mechanism.

TRPA1 (transient receptor potential ankyrin 1) is a non-selective, calcium-permeable cation channel that mediates pain by detecting environmental irritants and thermal stimuli. Although the role of TRPA1 in modulating pain perception is relatively well established, so far only a few human TRPA1 variants (N855S and A172V) have been associated with inherited neuropathic pain disorders. Here, we describe a novel TRPA1 variant (p. M978V) identified in two human subjects presenting with painful sensory neuropathy. Electrophysiological recordings demonstrate that the M978V variant confers gain-of-function properties to the TRPA1 channel, especially in response to allyl isothiocyanate (AITC; mustard oil), a well-characterised TRPA1 agonist. The M978V substitution enhances current density and shifts the half-maximal activation potential, rendering the channel more readily activated by electrophilic agonists, such as AITC. Furthermore, the mutant channel exhibits increased plasma membrane expression following AITC stimulation, suggesting that this single amino acid substitution affects both channel gating and trafficking. Using all-atom molecular dynamics simulation (MD), we highlighted that the variant is adjacent to the PIP2 binding site on the TRPA1 channel. We further show that depletion of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) increases current density in both WT and M978V channels. Importantly, the gain-of-function phenotype conferred by the M978V variant in response to AITC is dependent on the presence of PIP2. Collectively, our findings provide further evidence supporting the role of TRPA1 in human painful channelopathies and identify a previously unrecognised PIP2-dependent mechanism that regulates TRPA1 gain-of-function. Significance StatementIn this study we characterised the mechanism by which a rare TRPA1 variant leads to painful sensory neuropathy and discovered a novel modulatory PIP2-mediated regulation. Our in vitro data show that the variant confers gain-of-function properties to TRPA1 by enhancing its current density and open probability, as well as the channels surface membrane expression, in response to AITC, a known TRPA1 agonist. We also identified a novel interaction site for PIP2, a modulatory anionic lipid in the membrane of TRP channels. We have shown that abolishing endogenous PIP2 facilitates TRPA1 channel activation and that PIP2 is necessary for the variants gain-of-function properties, highlighting a new potential therapeutic avenue for neuropathic pain disorders.