Supporting Figures

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Fig. 5. NMM (20 mM) specifically activates calcium influx into HEK293 cells transfected with TRPA1, but not TRPV1, TRPV2, and TRPM8, as assessed by Fura-2/AM ratiometric calcium imaging. As a positive control, a specific agonist for each channel was used as follows: AITC (20 mM) for TRPA1, capsaicin (1 mM) for TRPV1, 2-aminophenylborinate (2-APB, 300 mM) for TRPV2, and menthol (50 mM) for TRPM8. Representative images were taken from one of four trials of two independent transfections.

Fig. 6. NMM activation is specifically ablated in triple mutant TRPA1-3C in transfected HEK293 cells, as measured by Fura-2/AM ratiometric calcium imaging. Sensitivity to AITC (50 mM) and THC (400 mM) is retained. Representative images were taken from one of three trials from two transfections.

Fig. 7. Triple mutant hTRPA1-3C retains the capacity to couple to m1 muscarinic receptor. HEK293 cells cotransfected with TRPA1-3C and m1AChR were recorded in the whole-cell configuration. Holding currents at -60 mV increased upon stimulation with 100 mM ACh in the inward direction (robust currents observed in 3 of 10 patch-clamped cells). (Scale bars: 40 pA and 1 min.)

Fig. 8. Oocytes expressing triple mutant hTRPA1-3C are very weakly responsive to acrolein (a; 100 mM) and cinnamaldehyde (b; 400 mM) (n = 5 each). Response to AITC (200 m M) is used as a positive control. (Scale bars: 200 nA and 1 min.)

Fig. 9. Lysine 708 is not necessary for response to AITC in wild-type channel. (a) Representative trace from an oocyte injected with hTRPA1-K708R (n = 5). Channel function assessed by reversible activation by AITC (200 mM), irreversible activation by NMM (200 mM), blockade with ruthenium red (10 mM). (Scale bars: 100 nA and 1 min.) (b) Current-voltage relationships of hTRPA1-K708R responses to AITC (red trace) and NMM (blue trace) show characteristic outward rectification, as assessed by 1-sec voltage ramps.