Figure 2
Capsazepine (CPZ) activates heterologously expressed human TRPA1.
(A) CPZ (10 μM) evokes hTRPA1-mediated currents in transfected HEK293t cells. Note the complete inhibition of currents by the selective TRPA1 antagonist HC030031 (HC, 10 μM). (B) Representative ramp currents evoked in a hTRPA1-transfected HEK293 cell by 400 ms voltage ramps from −100 to +100 mV applied every 4 s. Each symbol represents the current amplitude at −80 mV (squares) and +80 mV (circles). The CPZ-induced current was strongly inhibited by HC. (C) Examples of individual ramp currents corresponding to the filled symbols and numbers in B. (D) CPZ (50 μM, 10 s) evokes large calcium transients in hTRPA1-HEK293 cells (black trace, n = 128). HC (20 μM; red trace, n = 209) and A-967079 (10 μM; blue trace, n = 140) completely inhibited the response to CPZ. Data represent means (straight lines) ± SEMs (dotted lines). (E) The activation of hTRPA1 by CPZ (10 μM) is concentration-dependent. Black trace: hTRPA1-HEK293 cells (n = 52) were stimulated by increasing concentrations of CPZ for 20 s at 3 min-intervals. Gray trace: untransfected HEK293 cells (n = 101) were subjected to the same CPZ concentrations. (F) Activation of hTRPA1 by CPZ (1 μM) involves three critical cysteines in the N-terminus of the channel. Black trace: response of n = 123 HEK293 cells expressing WT hTRPA1 to successive applications of CPZ (1 μM), carvacrol (100 μM) and allyl isothiocyanate (AITC, 50 μM). Gray trace: response of n = 165 HEK293 cells expressing the mutant hTRPA1-3C to the same sequence of stimuli. Note the substantial reduction in the response of the hTRPA1-3C mutant to CPZ and AITC, but not to carvacrol. (G) The difference in CPZ sensitivity between genotypes was abolished at 100 μM CPZ. (H) Activation of hTRPA1 by CPZ can be prevented by the scavenger N-acetyl cysteine (NAC). Black trace: in control experiments hTRPA1-HEK293 cells were challenged with CPZ (50 μM; 60 s; n = 74). Red trace: in a separate experiment cells were first exposed for 30 s to a combination of CPZ (50 μM) and NAC (15 mM), immediately followed by CPZ alone for another 30 s (n = 83).
