Identification of both GABAA receptors and voltage-activated Na(+) channels as molecular targets of anticonvulsant α-asarone

Abstract

Alpha (α)-asarone, a major effective component isolated from the Chinese medicinal herb Acorus tatarinowii, is clinically used as medication for treating epilepsy, cough, bronchitis, and asthma. In the present study, we demonstrated that α-asarone targets central nervous system GABAA receptor as well as voltage-gated Na(+) channels. Using whole-cell patch-clamp recording, α-asarone inhibited spontaneous firing of output neurons, mitral cells (MCs), in mouse olfactory bulb brain slice preparation and hyperpolarized the membrane potential of MCs. The inhibitory effect of α-asarone persisted in the presence of ionotropic glutamate receptor blockers but was eliminated after adding a GABAA receptor blocker, suggesting that GABAA receptors mediated the inhibition of MCs by α-asarone. This hypothesis was supported by the finding that α-asarone evoked an outward current, but did not influence inhibitory postsynaptic currents (IPSCs). In addition to inhibiting spontaneous firing, α-asarone also inhibited the Nav1.2 channel, a dominant rat brain Na(+) channel subtype. The effects of α-asarone on a defined Nav1.2 were characterized using transfected cells that stably expressed the Nav1.2 channel isoform. α-Asarone displayed strong tonic inhibition of Nav1.2 currents in a concentration- and membrane potential-dependent fashion. α-Asarone reduced channel availability in steady-state inactivation protocols by enhancing or stabilizing Na(+) channel inactivation. Both Na(+) channel blockade and activation of GABAA receptors provide a possible mechanism for the known anti-epileptic effects of α-asarone. It also suggests that α-asarone could benefit patients with cough possibly through inhibiting a Na(+) channel subtype to inhibit peripheral and/or central sensitization of cough reflexes.

Keywords: GABAA receptors; anticonvulsant; central nerve Nav1.2 channel; olfactory bulb; sensitization of cough reflexes; sodium channel blocker; α-asarone.

FIGURE 1

Chemical structure of α-asarone and methyleugenol (4-allyl-1,2-dimethoxybenzene).

FIGURE 2

(A–D) Tonic inhibition of α-asarone on Na_v1.2 channels. (A) The current traces of Na_v1.2 illustrate the current–voltage (I–V) relationship of the channel. (B) The I–V relationships in control and in the presence of various concentrations of asarone. The currents were elicited by stepping to various depolarized potentials (ranging from – 80 to +100 mV in 10-mV increments) for 9 ms, and then returning to the holding potential of –100 mV. Peak currents at each depolarized potential were measured. The data are from a representative cell. (C) Superimposed current traces recorded before (control), during the application of 20 μM, 500 μM, and 2 mM α-asarone. The currents were recorded from the same cell and were elicited by a 10-ms pulse to 0 mV from the -100 mV holding potential. (D) Concentration–response curves for the inhibition of Na⁺ currents by α-asarone at different holding potentials. The cells were held at –100 mV, and –60 mV, respectively, and stepped to 0 mV for 10 ms. The peak currents in the presence of asarone were normalized to the corresponding control peak currents, and then averaged. Each point was the mean ± SEM of 4–6 cells. The lines are best fits for data to the Hill equation: y=1-xⁿ/(K_dⁿ+xⁿ), where y is the fractional current, K_d is the apparent dissociation constant for asarone, and n is the Hill coefficient. K_d and n were estimated using a Marquadt non-linear least-squares routine.

FIGURE 3

α-asarone on voltage-dependent availability and activation of Na_v1.2 channels. The shift of inactivation curves of Na_v1.2 channels by 500 μM asarone (n = 5). The voltage dependence of steady-state inactivation (h_∞) was examined by applying 500-ms prepulse potentials from –120 to 10 mV in 10-mV increments from a holding potential of –100 mV before stepping to the test potential (0 mV) for 35 ms. The peak current (I) for each cell was normalized with respect to the first value measured at test potential (0 mV). Conductance–voltage relationship: from the peak Na⁺ currents obtained in Figures 2A,B, the Na⁺ conductance values (G) in the absence and presence of 500 μM α-asarone were calculated, normalized to the maximum in control, and plotted as a function of membrane potentials (V). The smooth curves through the data are drawn according to the equation: y = 1/1 - exp[(V - V_h)/k]. Where V = membrane potential, V_h= the prepulse potential where the current is half-maximal, and k = the slope factor.

FIGURE 4

α-asarone suppressed the spiking activity of MCs. Original recording from a representative MC. Bath application of α-asarone reduced the firing rate and hyperpolarized the membrane potential. The arrow indicates the initial level of the resting potential.

FIGURE 5

(A–C) A GABA_A receptor antagonist rather than AMPA/kainate or NMDA receptor antagonists blocked the α-asarone-induced inhibition of MCs. (A) Original recording obtained from a MC shows that α-asarone-induced suppression of neuronal firing recorded in a representative MC in the presence of iontropic glutamate receptor antagonists CNQX and D-AP5. (B) Current trace shows that α-asarone failed to evoke the inhibitory effects in the presence of fast synaptic blockers. (C) In gabazine, a GABA_A receptor antagonist, the firing rate remained the same. Current trace shows that gabazine eliminated the inhibitory effects of α-asarone.

FIGURE 6

α-Asarone induced outward currents in MCs. (A) Original recording from a MC illustrates an outward current induced by 20 μM α-asarone. Low-Cl^- pipette solution containing sodium channel blocker QX-314 was used, and holding potential is 0 mV. (B) α-Asarone induced an outward current in MC with high-Cl^- pipette solution containing QX-314. Holding potential is at -60 mV. sIPSCs appear as downward deflections. The dotted green lines indicate the zero-current baselines.