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 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.

Fig. 1.

Phylogenetic analysis reveals a critical role for the S5 domain in anesthetic activation. (A) Representative currents recorded from rTRPA1-expressing HEK293 cells during a 100-ms voltage ramp from −100 to +100 mV in response to isoflurane (0.9 mM), propofol (1 mM), or AITC (100 µM, black trace). The baseline current in the absence of agonists was subtracted. (B) Pungent general anesthetics activate rat and human TRPA1 but not Drosophila. Mean current measured at +100 mV induced by isoflurane (0.9 mM) and propofol (1 mM) from HEK293 cells expressing rat, human, and Drosophila TRPA1. Data are mean ± SEM from 3 to 10 cells. (C) Schematic of WT dTRPA1, WT mTRPA1, dTRPA1-mN, mTRPA1-dTM5 receptors. (D) Current-voltage relationship for responses to isoflurane (Iso, blue traces) and AITC (black traces) in HEK293 cells transfected with chimeric TRPA1 channels shown in C. Note that baseline currents were subtracted.

Fig. 2.

S876 in TRPA1 S5 domain is critical for sensing isoflurane. (A) Alignment of S5 region used in chimeras from mouse, human, and Drosophila TRPA1. Amino acids in dTRPA1 divergent from the mammalian sequence are marked in yellow. (B) Representative I–V traces (−100 to +100 mV) evoked by isoflurane (0.9 mM, blue traces) and AITC (100 µM, black traces) for WT and mutant (S876V, T877L, and S876V/T877L) rTRPA1. (C and D) Mean currents evoked by isoflurane and propofol (1 mM) measured at +100 mV and −100 mV in HEK293 cells expressing WT and mutant rTRPA1. Data are mean ± SEM from three to six cells.

Fig. S1.

AITC sensitivity in rat WT TRPA1 and S876V mutants. Currents were recorded at −100 mV (n = 3–4). The smooth lines are best-fits to a Hill function.

Fig. 3.

Isoflurane inhibits A-967079 antagonism in a concentration-dependent manner. Representative current-voltage traces for rTRPA1 activated by AITC in the presence or absence of isoflurane (0.9 mM) (A) and in the presence of A-967079 (2 µM) (B). (C) Mean current (% of AITC alone) measured at +200 mV induced by AITC plus isoflurane and/or A-967079 (n = 6–9). Isoflurane relieves the inhibitory effect of A-967079. (D and E) Inhibition of isoflurane-evoked currents by A-967079 or HC-030031 (both 2 µM) measured at −100 and +100 mV (n= 4–5; *P < 0.01 compared with response to HC-030031).

Fig. 4.

Introduction of the A-967079 binding “pocket” into dTRPA1 confers sensitivity to isoflurane. (A) Alignment of S5, S6, and the first pore helix from rat and Drosophila TRPA1. Amino acids implicated in the sensitivity of rTRPA1 to the potent antagonist A-967079 are labeled yellow, and nonidentical residues in dTRPA1 are marked red. (B and C) AITC (500 µM)-evoked currents in WT and V933S/L934T/I941L/M1055V mutant dTRPA1 in the presence of 10 µM A-961079 (n = 6) or 20 µM HC-030031 (n = 4). (D and E) Representative I–V traces in response to isoflurane (0.9 mM, blue traces) and AITC (500 µM, black traces) obtained from voltage ramps for cells expressing WT and V933S/L934T/I941L/M1055V dTRPA1. (F) Mean current responses of isoflurane (measured at +100 mV, blue columns) in HEK293 cells expressing WT dTRPA1 (n = 8) and mutants (n = 5, 23, 3, and 6 for L934, V933S/L934T, V933S/L934T/I941L, and V933S/L934T/I941L/M1055V, respectively).

Fig. 5.

Molecular modeling and functional analysis identify TRPA1 residues involved in binding isoflurane and propofol. (A–C) Binding modes of isoflurane (A), propofol (B), and A-967078 (C) in human TRPA1 revealed by molecular docking. Note that Met-912 and Met-953 interact with isoflurane and propofol but not A-967078. (D) Mean currents (measured at +100 mV) evoked by isoflurane (0.9 mM), propofol (1 mM), or menthol (1 mM) in HEK293 cells expressing WT (n = 3) or mutant (n = 6–11) rTRPA1s. (E) Inhibition of AITC responses by A-967079 in WT (n = 5) and mutant (n = 4–6) rTRPA1s.