Figure 7. Studies with HEK-293 cells transfected with recombinant TRPA1.

(a,b) HEK-293 cell monolayers transfected with wild-type (WT) rat TRPA1 cDNA, but not a negative control empty vector (EV), exhibited an increase of [Ca²⁺]_i in response to JWU-A021 (1 μM), and this action of JWU-A021 was abrogated by the TRPA1 channel blocker A967079. (c,d) HEK-293 cells transfected with WT rat TRPA1 cDNA, but not a negative control EV, exhibited an increase of [Ca²⁺]_i in response to AITC (10 μM), and this action of AITC was abrogated by the TRPA1 channel blocker A967079. This experiment confirmed the expected failure of HEK-293 cells to express endogenous TRPA1 channels. (e) A concentration-dependent action of JWU-A021 to increase [Ca²⁺]_i was measured in HEK-293 cell monolayers transfected with wild-type (WT) rat TRPA1 cDNA. (f) HEK-293 cell monolayers transfected with mutant C622S rat TRPA1 cDNA responded to JWU-A021 in a manner nearly identical to that of cells transfected with WT TRPA1 (compare panels e,f). Thus, the non-electrophile JWU-A021 acted independently of C622 covalent modification. (g,h) The TRPA1 activator AITC stimulated an increase of [Ca²⁺]_i in HEK-293 cell monolayers transfected with WT rat TRPA1, and this action of AITC to increase [Ca²⁺]_i was greatly diminished in HEK-293 cells transfected with mutant C622S TRPA1 cDNA (compare panels g,h). Thus, the electrophile AITC must covalently modify C622 in order to fully activate the channel. For all examples depicted here, the findings are representative of a single experiment repeated a minimum of three times on three different occasions with similar results.