Figure 3
Capsazepine (CPZ) activates murine TRPA1 in dorsal root ganglion neurons.
(A) CPZ (50 μM) activates a subpopulation of AITC (100 μM)-sensitive mouse dorsal root ganglion (DRG) neurons. Individual responses from three different AITC- and capsaicin (CAP, 1 μM)-sensitive DRG neurons that were activated by CPZ. CPZ was applied for 20 s, AITC for 30 s and CAP for 10 s at intervals of 4 min allowing recovery. KCl (60 mM) was applied at the end of the experiment to to ensure viability of cultured neurons. (B) Averaged response of n = 135 AITC-sensitive neurons to three different concentrations of CPZ (25, 50, 100 μM, 20 s each). Note the concentration-dependence of the amplitude of Ca2+ transients. Straight traces represent mean and dotted traces represent SEMs. (C) Concentration-dependent increase of CPZ-induced [Ca2+]i in AITC-sensitive DRG neurons normalized to a depolarizing stimulus with KCl (60 mM). The EC50 of ∼30 μM was calculated by fitting to the Dose-Response function. Data are representative of two sets of experiments and show means ± SEM; for CPZ 1, 10, 25 μM: n = 169, for CPZ 25, 50, 100 μM: n = 138. (D) Illustrating populations of CPZ-, AITC- and CAP-sensitive DRG neurons and their overlap. In a total of 906 imaged neurons (selected by their response to KCl), 200 (22%) were activated by CPZ (50 μM), 301 (33%) by AITC (100 μM) and 323 (36%) by CAP (1 μM) (detailed analysis of fractions, see SI Figure Legend 3). (E) CPZ (100 μM, 20 s) activates AITC (100 μM, 20 s)-sensitive DRG neurons from TRPV1-deficient mice. Individual responses from different AITC-sensitive neurons that were activated by CPZ. About 90% (509/572 cells) of the TRPV1-deficient DRG neurons that were AITC-sensitive were responsive to CPZ. CAP (1 μM, 10 s) did not induce Ca2+ transients in TRPV1−/− neurons. (F) Lack of CPZ (100 μM)-induced Ca2+ influx in TRPA1-deficient DRG neurons. Individual responses from different CAP (1 μM)-sensitive DRG neurons (out of 448 neurons tested) that were neither activated by CPZ nor AITC (10 μM).
