Fig. 3.
QX-314 activates human (h) and rat (r) transient receptor potential vanilloid 1 (TRPV1) via the vanilloid-binding domain. (A and B) Mean responses of all measured cells with 5 mM (A, green solid line) and 30 mM (B, blue solid line) in cells expressing hTRPV1. Original traces from representative cells as displayed by gray lines, and the mean response of untransfected human embryonic kidney 293 (HEK-293) cells are displayed by the black solid line in A; 1 μM capsaicin was applied to verify the expression of hTRPV1. (C) Bar diagrams of the area under the curve (AUC) of fluorescence signals during the 60-s-long application of QX-314. (D) Bar diagram of the percentage of capsaicin-sensitive cells responding to QX-314 (***P < 0.001, ANOVA with Tukey post hoc test for AUC; and #P ≤ 0.006, Mann– Whitney U test). (E, G, I, and K) Representative traces of QX-314–induced membrane currents in cells expressing (E) hTRPV1, (G) rTRPV1, (I) rabbit (o)TRPV1, and (K) the o/rTRPV1-chimera. Test solutions are listed in the panel. Inset between E and I: the recording protocol; currents were recorded during 500-ms voltage ramps from −100 to 100 mV in cells held at −60 mV. Capsaicin was applied at the end for each experiment as functional control for TRPV1 expression. Although capsaicin was usually applied 1 μM, the capsaicin-insensitive oTRPV1 required 10 μM capsaicin to generate robust membrane currents. Note that no membrane currents were activated upon coapplication of QX-314 with 100 nM 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (BCTC) (E). (F, H, J, and L) Bar diagrams of averaged (mean ± SD) peak amplitudes of membrane currents determined at −100 or 100 mV during application of different test solutions. Note that similar to capsaicin, QX-314–induced currents displayed a pronounced outward rectification, resulting in large outward currents at 100 mV. Also, note the 10-fold increase in y-axis scale in J, showing that oTRPV1 is not activated by QX-314. Data are mean ± SD, ratio of fluorescence intensity (F 340/380 nm).