Identification of a putative binding site critical for general anesthetic activation of TRPA1

Abstract

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABA A 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 halogenbond 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.

Sites Contributing to TRPA1 Activation by the Anesthetic Propofol Identified by Photoaffinity Labeling

“…Both propofol and AziPm significantly activated mTRPA1 channels within concentration ranges similar to those used for propofol in previous reports (see Fig. S1) (7,9,12). The responses to both propofol and AziPm were specific to mTRPA1, as neither caused calcium influx in rat TRP vanilloid 1-expressing or empty vector, baculovirus-infected Sf21 cells (see Fig.…”

“…S6 and S5 form the channel gate within 6TM-channels; therefore it is not surprising that ligand binding near S6 influences the dynamics and activity of 6TM-channels (20,21). Interestingly, these residues do not share identity within the propofol-insensitive Drosophila TRPA1 (9,12), again suggesting that these residues may contribute to functionally relevant propofol-binding site(s) (Fig. 2 D).…”

“…Uncovering the propofol modulatory site(s) will further our understanding of the TRPA1 molecular machinery and physiological functions, as well as provide information for drug design. Ton et al (12) identified a series of mutations (S876V, S876V/T877L, M915L, and M956A/V) that eradicated propofol activation of rat TRPA1. These studies suggested a binding cavity, common with the TRPA1 antagonist A-967079 site (12,13), as an allosteric site for propofol modulation.…”

“…Ton et al (12) identified a series of mutations (S876V, S876V/T877L, M915L, and M956A/V) that eradicated propofol activation of rat TRPA1. These studies suggested a binding cavity, common with the TRPA1 antagonist A-967079 site (12,13), as an allosteric site for propofol modulation. However, site-directed mutagenesis cannot definitively distinguish ligand-binding sites from confounding alterations in the protein's dynamics.…”

Sevoflurane Increases Locomotion Activity in Mice

“…The mouse activity was recorded before (5 minutes in air as baseline tracks), during (30 minutes) and after the sevoflurane anesthesia (40-minutes recovery). Here, we used 1, 1.5- and 2-fold minimum alveolar concentrations (MAC), which correspond to 2, 3, and 4% of sevoflurane respectively at 37° C 1,3,15 . The total walking distance (in cm) and velocity (in cm/min) of movement were measured and quantified as indexes of hyperactivity.…”