Figure 6.
Camphor and menthol inhibit KV7.2/KV7.3 channels. A, Sample trace of KV7.2 in response to the indicated voltage step protocol showing block by camphor and washout. B, Outward currents mediated by KV7.2/KV7.3 channels of HEK293T cells were dose dependently and reversibly inhibited by increasing concentrations of menthol (3–1000 μm). Holding potential, 0 mV. C, Blocking effect of camphor (red) on different K+ channels depicted as fitted average. KV7.2/3 (red circles): IC50 = 1.42 ± 0.20 mm; minimum, 15.18 ± 3.77%; Hill coefficient, 0.88 ± 0.07 (n = 9). KV7.2 (down-pointing triangles): IC50 = 0.53 ± 0.11 mm; minimum, 2.76 ± 1.56%; Hill coefficient, 0.70 ± 0.02 (n = 12). KV7.3 (up-pointing triangles) was least affected by camphor (n = 4). TRESK (rhombs): IC50 = 10.62 ± 3.62 mm; minimum, 11.35 ± 4.65%; Hill coefficient, 0.79 ± 0.07 (n = 9). Relative inhibition of KV7.2/KV7.3 outward current by menthol (blue circles) was plotted as a function of menthol concentration. A logistic equation was fitted to the dose–response data, yielding an EC50 of 298 μm (n ≥ 10 for each data point). D, Menthol (50 μm) had synergistic effects on TRPM8 and KV7.2/KV7.3 channels cotransfected in HEK293T cells. At each command potential, a brief pulse of menthol (50 μm) was spritzed onto the recorded cells. Menthol-induced inward currents resulted predominantly from TRPM8 activation and KV7.2/KV7.3 inhibition at hyperpolarized and depolarized potentials, respectively. E, I–V relationships were calculated from experiments of D immediately before menthol application (black circles) and during peak menthol effect (open circles). Currents were normalized to their maximal amplitude before spritzing menthol (n = 9). All error bars indicate SEM.