The Transient Receptor Potential A1 channel (TRPA1) is involved in the transduction of inflammation-induced noxious stimuli from the periphery. Prior studies have characterized the properties of TRPA1 in heterologous expression systems. However, there is little information on the properties of TRPA1-mediated currents in sensory neurons. A capsaicin-sensitive subset of trigeminal ganglion (TG) sensory neurons was activated with TRPA1-specific agonists, mustard oil and the cannabinoid, WIN55,212. Mustard oil-and WIN55,212-gated currents exhibited marked variability in their kinetics of activation and acute desensitization. TRPA1-mediated responses in neurons also possess a characteristic voltage-dependency with profound outward rectification that is influenced by extracellular Ca 2+ and the type and concentration of TRPA1-specific agonists. Examination of TRPA1-mediated responses in a TRPA1-containing cells indicated that the features of neuronal TRPA1 are not duplicated in cells expressing only TRPA1, and instead, can be restored only when TRPA1 and TRPV1 channels are co-expressed. In summary, our results suggest that TRPA1-mediated responses in sensory neurons have distinct characteristics that can be accounted for by the co-expression of the TRPV1 and TRPA1 channels.

Unique features of sensory neuron subtypes are manifest by their distinct physiological and pathophysiological functions. Using patch-clamp electrophysiology, Ca 2+ imaging, calcitonin gene-related peptide release assay from tissues, protein biochemistry approaches, and behavioral physiology on pain models, this study demonstrates the diversity of sensory neuron pathophysiology is due in part to subtype-dependent sensitization of TRPV1 and TRPA1. Differential sensitization is influenced by distinct expression of inflammatory mediators, such as prostaglandin E 2 (PGE 2 ), bradykinin (BK), and nerve growth factor (NGF) as well as multiple kinases, including protein kinase A (PKA) and C (PKC). However, the co-expression and interaction of TRPA1 with TRPV1 proved to be the most critical for differential sensitization of sensory neurons. We identified N-and C-terminal domains on TRPV1 responsible for TRPA1-TRPV1 (A1-V1) complex formation. Ablation of A1-V1 complex with dominant-negative peptides against these domains substantially reduced the sensitization of TRPA1, as well as BK-and CFA-induced hypersensitivity. These data indicate that often occurring TRP channel complexes regulate diversity in neuronal sensitization and may provide a therapeutic target for many neuroinflammatory pain conditions. K E Y W O R D Spain, sensitization, sensory neurons, TRPA1, TRPV1 288 | PATIL eT AL.

Cannabinoid receptor antagonists have been utilized extensively in vivo as well as in vitro, but their selectivity has not been fully examined. We investigated activation of sensory neurons by two cannabinoid antagonists – AM251 and AM630. AM251 and AM630 activated trigeminal (TG) sensory neurons in a concentration-dependent fashion (threshold 1 μM). AM251 and AM630 responses are mediated by the TRPA1 channel in a majority (90–95%) of small-to-medium TG sensory neurons. AM630 (1–100 μM), but not AM251, was a significantly more potent agonist in cells co-expressing both TRPA1 and TRPV1 channels. We next evaluated AM630 and AM251 effects on TRPV1- and TRPA1-mediated responses in TG neurons. Capsaicin (CAP) effects were inhibited by pre-treatment with AM630, but not AM251. Mustard oil (MO) and WIN55,212-2 (WIN) TRPA1 mediated responses were also inhibited by pre-treatment with AM630, but not AM251 (25uM each). Co-treatment of neurons with WIN and either AM630 or AM251 had opposite effects: AM630 sensitized WIN responses, whereas AM251 inhibited WIN responses. WIN-induced inhibition of CAP responses in sensory neurons was reversed by AM630 pre-treatment and AM251 co-treatment (25μM each), as these conditions inhibit WIN responses. Hindpaw injections of AM630 and AM251 did not produce nocifensive behaviors. However, both compounds modulated CAP-induced thermal hyperalgesia in wild-type mice and rats, but not TRPA1 null-mutant mice. AMs also partially regulate WIN inhibition of CAP-induced thermal hyperalgesia in a TRPA1-dependent fashion. In summary, these findings demonstrate alternative targets for the cannabinoid antagonists, AM251 and AM630, in peripheral antihyperalgesia which involve certain TRP channels.