FIGURE 2. TRPA1 agonist binds to reactive cysteines, three of which are required for normal channel function.

a, Schematic of the method used to identify sites covalently bound by IA during on-cell treatment. b, Cartoon representation of TRPA1 showing each cysteine residue as a circle. c, MTSEA-biotin activates TRPA1 when applied in the intracellular recording solution in whole-cell configuration. Left panel, whole-cell currents at  120mV; right panel, instantaneous TRPA1 current–voltage relationships at points indicated in left panel. d, e, Voltage-gated whole-cell currents mediated by TRPA1 are significantly attenuated in three cysteine mutants: C415S, C422S and C622S. d, Voltage-evoked currents (left) and tail current analysis (right) of a representative wild-type (WT) TRPA1-expressing cell. e, Steady-state current density evoked by voltage steps is shown for the indicated constructs (mean  s.e.m., n = 17–23). All values for the cysteine mutants are significantly different from wild type (P < 0.005) and all values with the exception of C415S at +100 mV are significantly different from vector controls (P < 0.05). f, g, Whole-cell currents elicited by application of 30 M MO and 100 M icilin and normalized to leak-subtracted currents evoked by a step to +180 mV. Plotted is the ratio standard error for the number of individual determinations (cells) shown. *P < 0.05; **P < 0.01; ***P < 0.005 (Student's t-test).