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. 2013 Oct 16;33(42):16627–16641. doi: 10.1523/JNEUROSCI.1473-13.2013

Figure 3.

Figure 3.

Camphor exerts mixed agonist and antagonist effects on recombinant rTRPM8. A, B, Average response of HEK293T cells (n = 100) transiently transfected with rTRPM8 and submitted to repetitive short (three consecutive applications of 15 s each, numbered 1–3; A) and one longer (5 min followed by 15 s) stimulation with camphor (2 mm; red) or menthol (100 μm; blue; B); bar charts on the right illustrate the area under the curve (AUC) of consecutive responses. Menthol was applied after camphor applications to test for heterologous desensitization (red, but not blue lines). Black lines, Control menthol responses. Note that long application of camphor-induced potent homologous but not heterologous desensitization. C, Cold-activated inward currents in HEK293T cells transiently transfected with rTRPM8. Camphor applied alone or in combination with a cold stimulus had no effect on the cold-induced current through TRPM8. Icold through TRPM8 reached an average peak amplitude of 273 ± 73 pA (n = 8; cold-stimulus to ∼10°C) and of 304 ± 78 pA in the presence of camphor (n = 8; p > 0.05, Student's paired t test). Right trace, TRPM8 activation at 24°C displays a strong outward rectification during voltage ramps (500 ms) from −100 to +150 mV (lower trace); the effect of camphor 2 mm was very small. D, The 2 mm camphor partially blocked cold-activated currents of heterologously expressed TRPM8 when sensitized by 50 μm menthol. The camphor-induced inhibition of the cold-activated and menthol-sensitized peak current reached ∼60% (n = 6). E, Concentration–response relationship of camphor (EC50, 1.1 mm) and menthol (EC50, 0.027 mm) in HEK293T cells transiently transfected with rTRPM8 using conventional calcium imaging; asterisk signifies p < 0.001 using unpaired t test. F, Effect of camphor on cold responses (2 min) of cold- and menthol-sensitive (me) cultured mouse DRG neurons loaded with Fura-2AM. Camphor applied after a control cold stimulus reduced the cold-activated calcium signal of the second cold stimulus by 53% (red line; mean of n = 14, p = 0.006, Wilcoxon test). Repeated superfusion with cooled extracellular solution reduced the calcium signal of the second cold stimulus in menthol-sensitive cells by 16% (dotted black line; mean of n = 8 cells, p = 0.025, Wilcoxon test). Bar chart (right) compares AUC for cold-activated calcium signal of first and second cold responses. Red arrowhead, Some of the cells showed a rapid and transient calcium increase upon camphor application.