The Modulation of Ubiquinone, a Lipid Antioxidant, on Neuronal Voltage-Gated Sodium Current

Abstract

Ubiquinone, composed of a 1,4-benzoquinone and naturally produced in the body, actively participates in the mitochondrial redox reaction and functions as an endogenous lipid antioxidant, protecting against peroxidation in the pituitary-dependent hormonal system. However, the questions of if and how ubiquinone directly affects neuronal ionic currents remain largely unsettled. We investigated its effects on ionic currents in pituitary neurons (GH3 and MMQ cells) with the aid of patch-clamp technology. Ubiquinone decreased the peak amplitude of the voltage-gated Na⁺ current (I_Na) with a slowing of the inactivation rate. Neither menadione nor superoxide dismutase modified the ubiquinone-induced I_Na inhibition. In response to an isosceles-triangular ramp pulse, the persistent I_Na (I_Na(P)) at high- and low- threshold potentials occurred concurrently with a figure-eight hysteresis loop. With ubiquinone, the I_Na(P) increased with no change in the intersection voltage, and the magnitude of the voltage-dependent hysteresis of the current was enhanced. Ubiquinone was ineffective in modifying the gating of hyperpolarization-activated cation currents. In MMQ lactotrophs, ubiquinone effectively decreased the amplitude of the I_Na and the current inactivation rate. In sum, the effects of ubiquinone demonstrated herein occur upstream of its effects on mitochondrial redox processes, involved in its modulation of sodium channels and neuronal excitability.

Keywords: coenzyme Q10; current kinetics; persistent Na+ current; ubiquinone; voltage hysteresis; voltage-gated Na+ current.

Figure 1

Effects of Ubiquinone (CoQ10) on voltage-gated Na⁺ current (I_Na) measured from GH₃ pituitary cells. In these experiments, we kept cells immersed in Ca²⁺free Tyrode’s solution containing 10 mM of TEA, and the recording pipette was filled with a Cs⁺-containing solution. The composition of these solutions is detailed in the Materials and Methods section. (A) Representative I_Na traces taken (a) during the control period and during cell exposure (b) to 1 μM of CoQ10 or (c) to 3 μM of CoQ10. The right side of panel (A) indicates an expanded record taken from the dashed box on the left side, while the upper part shows the voltage-clamp profile used. The smooth gray line denotes the double exponential (i.e., the fast and slow components) fit that has been overlaid on the curve representing the current (open symbols). The summary bar graphs shown in (B), orange bars and (C), yellow bars illustrate the effects of CoQ10 (1 and 3 μM) on both the amplitude and the slow component of the inactivation time constant (τ_inact(S)), respectively, of the I_Na evoked in response to brief depolarizing voltage steps. Both the amplitude (i.e., at the beginning of voltage pulse) and the inactivation time constant (i.e., τ_inact(S) of the current with and without the addition of CoQ10) were measured when the GH₃ cells were depolarized from −80 to −10 mV in a period of 40 ms. Each point represents the mean ± SEM (n = 8). * Significantly different from the control group (p < 0.05) and ** significantly different from the addition of CoQ10 (1 μM) alone group (p < 0.05). (D) The concentration-dependent inhibition of the peak I_Na produced by CoQ10. Each cell was voltage-clamped at −80 mV, and the current amplitude recorded during cell exposure to different concentrations of CoQ10 was measured at the start of a 40 ms-long depolarizing voltage step to −10 mV. The values of IC₅₀, n_H, and E_max related to the CoQ10-induced inhibition of the peak I_Na were estimated to be 5.6 μM, 1.2, and 97%, respectively. The continuous sigmoidal curve, over which the data points were overlaid, demonstrates the best fit to the modified equation indicated in the Materials and Methods section. (E) Steady-state current versus voltage (I–V) relationships of peak I_Na obtained in the absence (■) and presence (○) of 3 μM CoQ10 (mean ± SEM; n = 7 for each point). Peak current amplitude was measured at the start of each depolarizing voltage step.

Figure 1

Effects of Ubiquinone (CoQ10) on voltage-gated Na⁺ current (I_Na) measured from GH₃ pituitary cells. In these experiments, we kept cells immersed in Ca²⁺free Tyrode’s solution containing 10 mM of TEA, and the recording pipette was filled with a Cs⁺-containing solution. The composition of these solutions is detailed in the Materials and Methods section. (A) Representative I_Na traces taken (a) during the control period and during cell exposure (b) to 1 μM of CoQ10 or (c) to 3 μM of CoQ10. The right side of panel (A) indicates an expanded record taken from the dashed box on the left side, while the upper part shows the voltage-clamp profile used. The smooth gray line denotes the double exponential (i.e., the fast and slow components) fit that has been overlaid on the curve representing the current (open symbols). The summary bar graphs shown in (B), orange bars and (C), yellow bars illustrate the effects of CoQ10 (1 and 3 μM) on both the amplitude and the slow component of the inactivation time constant (τ_inact(S)), respectively, of the I_Na evoked in response to brief depolarizing voltage steps. Both the amplitude (i.e., at the beginning of voltage pulse) and the inactivation time constant (i.e., τ_inact(S) of the current with and without the addition of CoQ10) were measured when the GH₃ cells were depolarized from −80 to −10 mV in a period of 40 ms. Each point represents the mean ± SEM (n = 8). * Significantly different from the control group (p < 0.05) and ** significantly different from the addition of CoQ10 (1 μM) alone group (p < 0.05). (D) The concentration-dependent inhibition of the peak I_Na produced by CoQ10. Each cell was voltage-clamped at −80 mV, and the current amplitude recorded during cell exposure to different concentrations of CoQ10 was measured at the start of a 40 ms-long depolarizing voltage step to −10 mV. The values of IC₅₀, n_H, and E_max related to the CoQ10-induced inhibition of the peak I_Na were estimated to be 5.6 μM, 1.2, and 97%, respectively. The continuous sigmoidal curve, over which the data points were overlaid, demonstrates the best fit to the modified equation indicated in the Materials and Methods section. (E) Steady-state current versus voltage (I–V) relationships of peak I_Na obtained in the absence (■) and presence (○) of 3 μM CoQ10 (mean ± SEM; n = 7 for each point). Peak current amplitude was measured at the start of each depolarizing voltage step.

Figure 2

Summary bar graph showing effects (i.e., percentage inhibition) of ubiquinone (CoQ10), CoQ10 plus menadione, CoQ10 plus superoxide dismutase, and coQ10 plus tefluthrin (tef) (mean ± SEM; n = 8 for each bar). The results reflect that CoQ10-mediated inhibition of I_Na is unlinked to the level of reactive oxygen species. Peak amplitudes of I_Na with or without different tested compounds were compared. * Significantly different from CoQ10 (3 μM) alone group (p < 0.05).

Figure 3

Effects of ubiquinone (CoQ10) on the voltage-dependent hysteresis of the persistent I_Na (I_Na(P)) in GH₃ cells. The cells were bathed in Ca²⁺-free Tyrode’s solution, and the recording pipette was filled with a Cs⁺-containing solution. When the whole-cell configuration was established, the examined cell was voltage-clamped at −50 mV, and then an upright isosceles-triangular ramp pulse was applied covering the voltage range from −100 to +50 mV for a duration of 200 ms (or a ramp speed of ±0.75 V/s). Panels (A,B) present representative current traces obtained during the control period and during cell exposure to 3 μM CoQ10, respectively. The inset in (A) shows the voltage-clamp protocol applied. The dashed lines in (A,B) indicate the direction of the trajectory of the I_Na(P) following the passage of time, indicating a figure-eight configuration of the current. * and ** denote the high-threshold I_Na(P) and the low-threshold I_Na(P), respectively, elicited by the ascending (upsloping, in blue color) and descending (downsloping, in red color) limb of a 400 ms-long isosceles-triangular ramp pulse. The summary bar graphs in (C,D) depict the effects of CoQ10 (3 and 10 μM) on the amplitude of the high-threshold I_Na(P) and the low-threshold I_Na(P), respectively, in the GH₃ cells (mean ± SEM; n = 8 for each bar). The amplitudes of the high-threshold I_Na(P) and the low-threshold I_Na(P) activated by a 200 ms-long isosceles-triangular ramp pulse were measured at −30 and −80 mV, respectively. * Significantly different from the control group (p < 0.05) and ** significantly different from the addition of CoQ10 (3 μM) alone group (p < 0.05).

Figure 4

Mild inhibitory effect of ubiquinone (CoQ10) on the erg-mediated K⁺ current (I_K(erg)) in GH₃ cells. In these experiments, the cells were immersed in a high-K⁺ (around 140 mM of K⁺), Ca²⁺-free solution, and the recording pipette was filled with a K⁺-containing internal solution. (A) Representative current traces obtained with (their lower part) and without (their upper part) the addition of 10 μM of CoQ10. The uppermost part denotes the voltage-clamp protocol applied to the examined cells. Of note, the potential traces shown in different colors correspond to the current traces activated in response to each hyperpolarizing voltage. (B,C) show the I–V relationships of the peak (■) and late (○) deactivating I_K(erg) obtained in the control period and during cell exposure to 10 μM of CoQ10, respectively. The current amplitude was recorded at the beginning (■) and the end (○) of each 1 s-long hyperpolarizing voltage pulse from a holding potential of −10 mV. Each point is the mean ± SEM (n = 8, for each point). The dashed box in (B) indicates the voltage-dependent inactivation of the I_K(erg) present in these cells, according to the steady-state I–V relationship of sustained I_K(erg).

Figure 5

Effects of ubiquinone (CoQ10) on the hyperpolarization-activated cation current (I_h) in GH₃ cells. The introduction of CoQ10 was ineffective at perturbing the amplitude and gating of the hyperpolarization-activated cation current (I_h) in GH₃ cells. This set of experiments was conducted in cells which were bathed in Ca²⁺-free Tyrode’s solution containing 1 μM of TTX, and the pipette was filled with a K⁺-containing solution. (A) Representative current traces obtained (a) during the control period and during cell exposure (b) to 10 μM of CoQ10 and (c) to 10 μM of CoQ10 plus 3 μM of IVA. The voltage-clamp protocol applied is illustrated in the upper part. (B) A summary bar graph showing the effects of CoQW10 and CoQ10 plus IVA on the amplitude of the I_h (mean ± SEM; n = 8, for each bar). The current amplitude was measured at the endpoint of the hyperpolarizing step pulse from −40 to −110 mV for a duration of 2 s. * Significantly different from the control group (p < 0.05).

Figure 6

Effects of ubiquinone (CoQ10) on the I_Na in MMQ pituitary cells. The whole-cell experiments were conducted with MMQ cells, which were kept in Ca²⁺-free Tyrode’s solution, and we backfilled the pipette with a Cs⁺-containing solution. (A) Representative current traces obtained (a) during the control period and during cell exposure (b) to 3 μM of CoQ10 and (c) to 3 μM of CoQ10 plus 10 μM of ran. The inset demonstrates the voltage-clamp protocol applied to the cell. (B) A summary bar graph showing the effects of CoQ10, CoQ10 plus ran, and CoQ10 plus gom A (mean ± SEM; n = 8). The current amplitude was measured at the beginning of the depolarizing pulse from −80 to −10 mV. * Significantly different from the control group (p < 0.05), and ** significantly different from the addition of CoQ10 (3 μM) alone group (p < 0.05).