Pungent And Non-Pungent General Anesthetics Interact With Trpa1 Via Distinct Binding Pocket In The Pore Domain Region

General anesthetics (GAs) suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile and intravenous GAs can excite peripheral sensory nerves to cause pain and irritation upon administration. Noxious GAs activate Transient Receptor Potential Ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms remain unknown. Here we show that in addition to activation by pungent GAs, a broad variety of noxious and non-noxious anesthetics inhibit agonist-evoked activation of mammalian but not drosophila TRPA1. Analysis of chimeric drosophila and mouse TRPA1 channels reveal that the 5th transmembrane domain (S5) is critical for anesthetic sensitivity. We identify two amino acids at the N-terminal S5 domain required for the inhibitory effects of GAs and introduction of these residues into dTRPA1 confers GA-induced block in these channels. Further, we show that activation by noxious GAs requires several amino acids in the S5, S6 and the first pore helix previously implicated in binding of the high-affinity antagonist, A-967079. Notably, isoflurane relieves A-967079-mediated inhibition consistent with a competitive interaction and in silico modeling predicts that pungent GAs occupy the A-967079 binding pocket. Thus inhibition and activation by GAs are governed by distinct amino acids in the pore domain region. These data reveal the mechanism by which TRPA1 discriminates between pungent and non-pungent GAs, and provide structural requirements for designing antagonists to counter the noxious properties of GAs.