Imiquimod enhances excitability of dorsal root ganglion neurons by inhibiting background (K(2P)) and voltage-gated (K(v)1.1 and K(v)1.2) potassium channels

Abstract

Background: Imiquimod (IQ) is known as an agonist of Toll-like receptor 7 (TLR7) and is widely used to treat various infectious skin diseases. However, it causes severe itching sensation as its side effect. The precise mechanism of how IQ causes itching sensation is unknown. A recent report suggested a molecular target of IQ as TLR7 expressed in dorsal root ganglion (DRG) neurons. However, we recently proposed a TLR7-independent mechanism, in which the activation of TLR7 is not required for the action of IQ in DRG neurons. To resolve this controversy regarding the involvement of TLR7 and to address the exact molecular identity of itching sensation by IQ, we investigated the possible molecular target of IQ in DRG neurons.

Findings: When IQ was applied to DRG neurons, we observed an increase in action potential (AP) duration and membrane resistance both in wild type and TLR7-deficient mice. Based on these results, we tested whether the treatment of IQ has an effect on the activity of K(+) channels, K(v)1.1 and K(v)1.2 (voltage-gated K(+) channels) and TREK1 and TRAAK (K(2P) channels). IQ effectively reduced the currents mediated by both K(+) channels in a dose-dependent manner, acting as an antagonist at TREK1 and TRAAK and as a partial antagonist at K(v)1.1 and K(v)1.2.

Conclusions: Our results demonstrate that IQ blocks the voltage-gated K(+) channels to increase AP duration and K(2P) channels to increase membrane resistance, which are critical for the membrane excitability of DRG neurons. Therefore, we propose that IQ enhances the excitability of DRG neurons by blocking multiple potassium channels and causing pruritus.

Figure 1

Effects of IQ on AP duration and membrane resistance of DRG neurons in WT and TLR7 KO mice. A. Representative trace of AP of DRG neuron from WT and TLR7 KO before and after application of IQ. Bar graph indicates the average of AP duration of DRG neurons with or without IQ treatment from WT and TLR7 KO mice. B. Left column shows the representative example of change of membrane resistance induced by injection of negative current. Average effect of IQ on membrane resistance compared with WT and KO is shown as bar graph. Protocol for AP induction and membrane resistance by current injection was shown in bottom of traces of A and C. The number in each bar indicates the number of neurons. Error bars indicate S.E.M. (**, P > 0.01).

Figure 2

Partial inhibitory effect of IQ on K_v1.1 and K_v1.2 activity. A, C Effect of different concentrations of IQ on K_v1.1 (A) and K_v1.2 (C) currents induced by a voltage step. Protocol of voltage step shown below the traces. B, D. Average % of remaining currents of K_v1.1 (B) and K_v1.2 (D) during IQ (1, 10, 100 and 500 μM) application. Concentration-response relationship was obtained by fitting with logistic function. IC₅₀ of IQ on K_v1.1 and K_v1.2 is 39.2 μM and 21.32 μM, respectively. Error bars indicate S.E.M.

Figure 3

Antagonistic effect of IQ on TREK1 and TRAAK, K_2P channel. A, C Inhibitory effect of IQ on TREK1 (A) and TRAAK (C) channels as shown by instant I-V relationship. B, D Average % of remaining currents at each concentration of IQ on TREK1 (B) and TRAAK (D) channels. Fitted line was used to obtain IC₅₀ (TREK1, 81.1 μM; TRAAK, 181.7 μM) of IQ on K_2P channels. Error bars indicate S.E.M.