General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.
Keywords: A-967079; TRPA1; general anesthetic; isoflurane; propofol.