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

Figure 5.

Figure 5.

Camphor blocks potassium channels. A, Top, Representative current-clamp recording from a cultured TRPM8-positive and cold-sensitive DRG neuron of a TRPM8EGFPf/+ mouse. Application of camphor did not induce action potential discharge, but caused partial reduction of the number of discharged action potentials during cooling (control, 116 spikes per cold stimulation; camphor, 77; wash, 106). The cold-activated voltage change and the temperature of activation were unchanged (22, 22.2, and 22.8°C, respectively). Middle trace, Temperature time course. Bottom, Expanded first and second cold response. B, Camphor reduced the cold response (action potentials discharge during the falling phase of the cold stimulus) in all TRPM8-expressing cells (n = 5; p = 0.04 Wilcoxon test). C, Camphor reversibly decreased rheobase current, resulting in an increased excitability (n = 5). Current injections in 10 pA steps and of a length of 1000 ms were applied (64 ± 9.8, 44 ± 9.3, and 70 ± 15 pA, respectively; n = 5, p = 0.04, Wilcoxon test). D, Potassium currents activated by depolarizing voltage pulses from −80 to +60 mV from a holding potential of −60 mV in a cultured DRG neuron. Sodium currents were blocked with 700 nm TTX. E, I–V relationship of potassium currents recorded in DRG neurons; the camphor-induced blocking effect could be washed out and reached ∼50% at positive voltages (p < 0.05, t test). F, HEK293T cells transiently expressing the K2P channels TRESK, TRAAK, TREK1, and TREK2, and KV7.2 and KV7.3 were loaded with membrane potential dye, and the relative change in fluorescence was measured using a FLIPR Tetra plate reader and is shown as change relative to baseline. G, Stimulation with camphor caused a concentration-dependent change in fluorescence in HEK293T cells transiently expressing TRESK (open diamonds; EC50 = 8.57 mm) or KV7.3 (open up-pointing triangles; EC50 = 9.38 mm). Cells expressing KV7.2 (open down-pointing triangles; EC50 = 1.92 mm) were most sensitive to camphor, while there was no effect in untransfected HEK cells (closed diamonds).