Phoneutria toxin PnTx3-5 inhibits TRPV1 channel with antinociceptive action in an orofacial pain model.

Capsaicin, an agonist of TRPV1, evokes intracellular [Ca2+] transients and glutamate release from perfused trigeminal ganglion. The spider toxin PnTx3-5, native or recombinant is more potent than the selective TRPV1 blocker SB-366791 with IC50 of 47 ± 0.18 nM, 45 ± 1.18 nM and 390 ± 5.1 nM in the same experimental conditions. PnTx3-5 is thus more potent than the selective TRPV1 blocker SB-366791. PnTx3-5 (40 nM) and SB-366791 (3 μM) also inhibited the capsaicin-induced increase in intracellular Ca2+ in HEK293 cells transfected with TRPV1 by 75 ± 16% and 84 ± 3.2%, respectively. In HEK293 cells transfected with TRPA1, cinnamaldehyde (30 μM) generated an increase in intracellular Ca2+ that was blocked by the TRPA1 antagonist HC-030031 (10 μM, 89% inhibition), but not by PnTx3-5 (40 nM), indicating selectivity of the toxin for TRPV1. In whole-cell patch-clamp experiments on HEK293 cells transfected with TRPV1, capsaicin (10 μM) generated inward currents that were blocked by SB-366791 and by both native and recombinant PnTx3-5 by 47 ± 1.4%; 54 ± 7.8% and 56 ± 9.0%, respectively. Intradermal injection of capsaicin into the rat left vibrissa induced nociceptive behavior that was blocked by pre-injection with either SB-366791 (3 nmol/site i.d., 83.3 ± 7.2% inhibition) or PnTx3-5 (100 fmol/site, 89 ± 8.4% inhibition). We conclude that both native and recombinant PnTx3-5 are potent TRPV1 receptor antagonists with antinociceptive action on pain behavior evoked by capsaicin.