Effects of Sesamin, the Major Furofuran Lignan of Sesame Oil, on the Amplitude and Gating of Voltage-Gated Na+ and K+ Currents

Sesamin (SSM) and sesamolin (SesA) are the two major furofuran lignans of sesame oil and they have been previously noticed to exert various biological actions. However, their modulatory actions on different types of ionic currents in electrically excitable cells remain largely unresolved. The present experiments were undertaken to explore the possible perturbations of SSM and SesA on different types of ionic currents, e.g., voltage-gated Na+ currents (INa), erg-mediated K+ currents (IK(erg)), M-type K+ currents (IK(M)), delayed-rectifier K+ currents (IK(DR)) and hyperpolarization-activated cation currents (Ih) identified from pituitary tumor (GH3) cells. The exposure to SSM or SesA depressed the transient and late components of INa with different potencies. The IC50 value of SSM needed to lessen the peak or sustained INa was calculated to be 7.2 or 0.6 μM, while that of SesA was 9.8 or 2.5 μM, respectively. The dissociation constant of SSM-perturbed inhibition on INa, based on the first-order reaction scheme, was measured to be 0.93 μM, a value very similar to the IC50 for its depressant action on sustained INa. The addition of SSM was also effective at suppressing the amplitude of resurgent INa. The addition of SSM could concentration-dependently inhibit the IK(M) amplitude with an IC50 value of 4.8 μM. SSM at a concentration of 30 μM could suppress the amplitude of IK(erg), while at 10 μM, it mildly decreased the IK(DR) amplitude. However, the addition of neither SSM (10 μM) nor SesA (10 μM) altered the amplitude or kinetics of Ih in response to long-lasting hyperpolarization. Additionally, in this study, a modified Markovian model designed for SCN8A-encoded (or NaV1.6) channels was implemented to evaluate the plausible modifications of SSM on the gating kinetics of NaV channels. The model demonstrated herein was well suited to predict that the SSM-mediated decrease in peak INa, followed by increased current inactivation, which could largely account for its favorable decrease in the probability of the open-blocked over open state of NaV channels. Collectively, our study provides evidence that highlights the notion that SSM or SesA could block multiple ion currents, such as INa and IK(M), and suggests that these actions are potentially important and may participate in the functional activities of various electrically excitable cells in vivo.


Introduction
Sesame seeds and sesame oil have been widely recognized as health foods in Asian countries [1,2]. In comparison with other edible oils extracted from diverse seeds, sesame oil is extremely stable, possibly due to the effective antioxidant activities presumably attributed to its abundance of lipid-soluble furofuran lignans, such as sesamin (SSM) and sesamolin (SesA) [3][4][5][6][7]. Emerging research has previously demonstrated that SSM and SesA, the two major furofuran lignans of sesame oil, are able to suppress lipid peroxidation in erythrocytes [8], to inhibit the intestinal absorption of cholesterol and hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity [9], to prevent chemically induced mammary cancer, to inhibit D 5 -desaturase and the chain elongation of C18 fatty acids [10], and to protect hypoxic neuronal and PC12 cells by suppressing ROS generation and MAPK activation [11][12][13], as well as to exhibit antihypertensive or cardioprotective effects [1,3,14]. Earlier reports have revealed that either probucol or the triterpenoid fraction of Ganoderma, known to possesses the antioxidant activity, could perturb the activity of ionic currents in pituitary lactotrophs [15,16]. However, whether these therapeutic lignans (e.g., SSM, SesA) can directly perturb the activity of membrane ion currents is largely uncertain.
Molecular studies of epileptogenesis have revealed that specific ion channels play essential roles in both genetic and acquired forms of epilepsy, particularly voltage-gated Na + (Na V ) channels [17][18][19][20][21]. Nine isoforms (Na V 1.1-1.9) are found in mammalian excitable tissues, including the central nervous system, peripheral nervous system, endocrine system, skeletal muscles, and heart [22]. Moreover, several inhibitors known to preferentially block the late component of voltage-gated Na+ currents (I Na ), such as ranolazine, eugenol, and perampanel, have been reported to suppress seizure activity [23][24][25]. However, whether SSM or SesA are capable of exerting any perturbation on the amplitude and gating of I Na in response to rapid membrane depolarization remains poorly understood, though SSM was previously noted to activate transient receptor potential vanilloid type 1 in endothelial cells [26]. Alternatively, the presence of SSM has been previously revealed to suppress damage or apoptosis by streptozotocin in endocrine cells [27][28][29].
For the reasons described above, the goal of the present study was to explore whether SSM and SesA could exert any perturbations on different types of ionic currents (e.g., I Na ) present in pituitary GH 3 cells. The biophysical and pharmacological properties of ionic currents, including voltage-gated I Na , resurgent I Na (I Na(R) ), M-type K + currents (I K(M) ), erg-mediated K + currents (I K(erg) ), delayed-rectifier K + currents (I K(DR) ) and hyperpolarization-activated cation currents (I), were extensively studied in these cells. Moreover, the present work aimed to use a mathematical modeling approach for the evaluation of the perturbating actions on Na V -channel kinetics caused by SSM. The findings from the present observations highlight the notion that the furofuran lignans, such as SSM and SesA, are capable of perturbing the amplitude of I Na effectively in a concentration-, time-, and state-dependent manner.

Inhibitory Effect of Sesamin (SSM) on Voltage-Gated Na + Currents (I Na ) Identified in GH 3 Cells
In an initial step of the experiments, we examined the effects of SSM on the amplitude and gating of I Na in response to rapid membrane depolarization. Cells were bathed in Ca 2+ -free Tyrode's solution containing 10 mM tetraethylammonium chloride (TEA) and the recording pipette was backfilled with a Cs + -containing solution. As illustrated in Figure 1A, after 1 min of exposing cells to 3 or 10 µM SSM, the amplitude in the peak and sustained component of I Na elicited by rapid membrane depolarization from −80 mV was evidently decreased. For example, when rapid membrane depolarization from −80 to −10 mV with a duration of 40 msec was delivered (indicated in the inset of Figure 1A) to evoke I Na [30], the addition of 3 µM SSM caused a decrease in the peak or sustained amplitude of I Na to 139 ± 11 pA (n = 11, P < 0.05) or 12 ± 3 pA (n = 11, P < 0.05), respectively, from the control values of 248 ± 18 or 21 ± 2 pA (n = 11). After the removal of SSM, the peak and sustained amplitude returned to 232 ± 16 or 19 ± 2 pA (n = 7, P < 0.05). . The values of IC50 and nH for SSM-induced inhibition of sustained INa were calculated to be 0.6 μM and 1.2, respectively, whereas those for peak INa were 7.2 μM and 1.2, respectively. The vertical dashed line indicates the IC50 value required for SSM-perturbed inhibition of peak or sustained INa amplitude. Of note, the SSM addition is capable of differentially and concentration-dependently decreasing the amplitude of peak and sustained INa in GH3 cells, without any modifications to the Hill coefficient of the curve. (C) Time-dependent block of INa inactivation caused by SSM in GH3 cells. The reciprocal of the time constant of the rate of block (i.e., τinact(S) −1 ) achieved by exponential fits of the slow component of INa inactivation (τinact(S)) was constructed and plotted against the different concentrations of SSM applied. Data points were well fitted to a linear regression, reflecting that SSM-perturbed blocking occurs with a molecularity of 1. (D) Average I-V relationship of peak INa achieved in the absence (■) and presence (○) of 3 μM SSM (mean ± SEM; n = 8 for each point). Of note, no conceivable modification in the overall I-V relationship of peak INa in the absence and presence of SSM was demonstrated in GH3 cells. The statistical analyses were made by ANOVA-2 for repeated measures, P(factor 1) < 0.05, P(factor 2) < 0.05, P(interaction) < 0.05, followed by Duncan's post hoc test, P < 0.05. * Significantly different from controls measured at the same level of membrane potential (P < 0.05). Figure 1B illustrates that the presence of SSM can concentration-dependently depress the amplitude of peak or sustained INa activated during rapid membrane depolarization. The IC50 value needed for the SSM-perturbed decrease of peak or sustained INa identified in GH3 cells was 7.2 or 0.6 μM, respectively, the value of which was noticed to be distinct significantly between its effects on The whole-cell recordings were undertaken in cells bathed in Ca 2+ -free Tyrode's solution containing 10 mM tetraethylammonium chloride (TEA) and the patch pipette was filled with K + -containing solution. (A) Representative I Na traces obtained in the absence (a) and presence of 3 µM SSM (b) or 10 µM SSM (c). Inset shows the voltage-clamp profile applied. (B) Concentration-dependent effects of SSM on the peak and sustained components of I Na . I Na was evoked by abrupt depolarization from −80 to −10 mV. Current amplitudes obtained during cell exposure to different concentrations (0.1-100 µM) of SSM were measured at the beginning ( , peak I Na ) and end ( , sustained I Na ), evoked by depolarizing voltage. Each point presented in the figure depicts the mean ± standard error of the mean (SEM, n = 9-12). The values of IC 50 and n H for SSM-induced inhibition of sustained I Na were calculated to be 0.6 µM and 1.2, respectively, whereas those for peak I Na were 7.2 µM and 1.2, respectively. The vertical dashed line indicates the IC 50 value required for SSM-perturbed inhibition of peak or sustained I Na amplitude. Of note, the SSM addition is capable of differentially and concentration-dependently decreasing the amplitude of peak and sustained I Na in GH 3 cells, without any modifications to the Hill coefficient of the curve. (C) Time-dependent block of I Na inactivation caused by SSM in GH 3 cells. The reciprocal of the time constant of the rate of block (i.e., τ inact(S) −1 ) achieved by exponential fits of the slow component of I Na inactivation (τ inact(S) ) was constructed and plotted against the different concentrations of SSM applied. Data points were well fitted to a linear regression, reflecting that SSM-perturbed blocking occurs with a molecularity of 1. (D) Average I-V relationship of peak I Na achieved in the absence ( ) and presence ( ) of 3 µM SSM (mean ± SEM; n = 8 for each point). Of note, no conceivable modification in the overall I-V relationship of peak I Na in the absence and presence of SSM was demonstrated in GH 3 cells. The statistical analyses were made by ANOVA-2 for repeated measures, P(factor 1) < 0.05, P(factor 2) < 0.05, P(interaction) < 0.05, followed by Duncan's post hoc test, P < 0.05. * Significantly different from controls measured at the same level of membrane potential (P < 0.05). Figure 1B illustrates that the presence of SSM can concentration-dependently depress the amplitude of peak or sustained I Na activated during rapid membrane depolarization. The IC 50 value needed for the SSM-perturbed decrease of peak or sustained I Na identified in GH 3 cells was 7.2 or 0.6 µM, respectively, the value of which was noticed to be distinct significantly between its effects on these two components. The obtained results thus demonstrate that SSM has a depressant action on the peak or sustained I Na functionally expressed in GH 3 cells.

Kinetic Constants of I Na Block by SSM
During cell exposure to SSM, the I Na in response to brief depolarization exhibited a decline in peak amplitude followed by a rise in the exponential decay of the current. For this reason, it would thus be critical to gain information about the kinetics of the SSM-induced block of these currents observed in these cells. The concentration dependence of I Na decay (i.e., current inactivation) during a brief depolarization caused by the presence of SSM was derived and is illustrated in Figure 1C. It is important to emphasize that the effect of SSM on I Na resulted in a concentration-dependent rise in the rate of current decay, as well as in a considerable decrease in the sustained current, notwithstanding its ineffectiveness in perturbing the initial activation phase of I Na responding to brief depolarizing pulse. In other words, increasing the SSM concentration not only caused a reduction in the peak amplitude of I Na , but also remarkably enhanced the inactivation rate of the current in response to abrupt membrane depolarization. It stands to reason, therefore, that the inhibitory effect of SSM on I Na identified from GH 3 cells can be reflected with a state-dependent blocker which binds favorably to the open state of the Na V channel according to a minimal binding scheme, given as follows: where α and β is the kinetic constant for the opening or closing of the Na V channel, k +1  Figure 1C), while SSM presence did not alter the fast component of the I Na inactivation time course. Because a Hill coefficient of approximately 1 was obtained from the concentration-response curve, the block or unblock rate constant achieved in this study was evaluated using the formula given as follows: In this formula, the parameter value of k +1 * (the slope) and k -1 (the intercept) was calculated.
As predicted from this minimum binding scheme, the relationship between 1/τ inact(S) and [SSM] became linear with a correlation coefficient of 0.97 ( Figure 1C). The resultant rate constant of blocking or unblocking perturbed by the addition of SSM was calculated to be 0.0449 msec −1 µM −1 or 0.0415 msec −1 , respectively; as a consequence, a value of 0.93 µM for the dissociation constant (K D = k −1 /k * +1 ·[SSM]) of SSM could be achieved.
We also further examined effects of SSM on peak I Na measured at different levels of membrane potential. As shown in Figure 1D, the experimental observations revealed that the overall current-voltage (I-V) relationship of peak I Na attained between the absence and presence of 3 µM SSM did not differ noticeably, though the peak amplitude of the current measured at the level of each voltage was significantly decreased in the presence of SSM. In another experiment, we tested the effects of SSM, sesamolin (SesA), SSM plus tefluthrin, and SSM plus telmisartan on the peak amplitude of I Na responding to rapid membrane depolarization to −10 mV from a holding potential of −80 mV. Tefluthrin, a type I pyrethroid insecticide, and telmisartan, a blocker of angiotensin II receptors, were previously demonstrated to activate I Na directly and effectively [20,[30][31][32][33]. As shown in Figure 2, SSM or SesA, at a concentration of 3 µM, produced inhibitory effects on the peak amplitude of I Na . Furthermore, in the continued presence of SSM (3 µM), the subsequent addition of either tefluthrin (10 µM) or telmisartan (10 µM) was effective in reversing the SSM-induced inhibition of peak I Na .

Concentration-Dependent Inhibition of INa Caused by Sesamolin (SesA)
The effects of SesA, another furofuran lignan, on INa in response to an abrupt depolarizing pulse were further examined and compared in this study. The concentration-dependent relationships among the inhibitory effects of SesA on the peak and sustained component of INa are illustrated in Figure 3. The IC50 value of SesA required for its effect on the peak or sustained INa measured at the beginning or end of a brief depolarizing pulse was calculated to be 9.8 and 2.5 μM, respectively, though these values were relatively higher than for the SSM used for the blocking of the peak or sustained INa in GH3 cells.

Figure 2.
Comparison among the effects of SSM, SesA, SSM plus tefluthrin (Tef), and SSM plus telmisartan (Tel) on the peak amplitude of I Na . Experiments were conducted to measure the I Na in cells which were immersed in Ca 2+ Tyrode's solution, and once the whole-cell configuration was achieved, cells were rapidly depolarized to −10 mV from a holding potential of −80 mV. Current amplitude was measured at the start of a brief depolarizing pulse. (A) Representative I Na traces obtained in the control (a, both sides), during cell exposure to 3 µM SSM (b, both sides), and then to 3 µM SSM plus 10 µM tefluthrin (left side) or 3 µM SSM plus 10 µM telmisartan (right side). The upper part denotes the voltage protocol applied. In (B), each bar represents the mean ± SEM (n = 9). The statistical analyses were done by ANOVA-1, P < 0.05, followed by Duncan's post hoc test, P < 0.05. Tef: tefluthrin; Tel: telmisartan. * Significantly different from control (P < 0.05) and † significantly different from the SSM (3 µM) alone group (P < 0.05).

Concentration-Dependent Inhibition of I Na Caused by Sesamolin (SesA)
The effects of SesA, another furofuran lignan, on I Na in response to an abrupt depolarizing pulse were further examined and compared in this study. The concentration-dependent relationships among the inhibitory effects of SesA on the peak and sustained component of I Na are illustrated in Figure 3. The IC 50 value of SesA required for its effect on the peak or sustained I Na measured at the beginning or end of a brief depolarizing pulse was calculated to be 9.8 and 2.5 µM, respectively, though these values were relatively higher than for the SSM used for the blocking of the peak or sustained I Na in GH 3 cells.

Inhibitory Effect of SSM on Resurgent I Na (I Na(R) ) in GH 3 Cells
We next wanted to determine whether SSM exerts any effects on I Na(R) identified from these cells [30]. The whole-cell experiments on I Na(R) were undertaken when each cell was voltage clamped at −80 mV and a brief depolarizing step to +20 mV was delivered to activate transient I Na . The I Na(R) upon repolarization to various potentials ranging between −50 and 0 mV was thereafter measured at the end of voltage pulses ( Figure 4). The effect of SSM on I Na(R) was examined at various membrane potentials, and the I-V relationship of I Na(R) with or without the addition of SSM was constructed. The presence of SSM (1 µM) was capable of decreasing I Na(R) with no noticeable change in its voltage dependence in GH 3 cells, since the overall shape of the I-V curves for I Na(R) appearing between the absence and presence of SSM appeared to be similar. For example, at the level of −30 mV, the exposure to 1 µM SSM resulted in a decrease in I Na(R) amplitude from 46 ± 6 to 22 ± 5 pA (n = 8, P < 0.05).

Inhibitory Effect of SSM on Resurgent INa (INa(R)) in GH3 Cells
We next wanted to determine whether SSM exerts any effects on INa(R) identified from these cells [30]. The whole-cell experiments on INa(R) were undertaken when each cell was voltage clamped at −80 mV and a brief depolarizing step to +20 mV was delivered to activate transient INa. The INa(R) upon repolarization to various potentials ranging between −50 and 0 mV was thereafter measured at the end of voltage pulses ( Figure 4). The effect of SSM on INa(R) was examined at various membrane potentials, and the I-V relationship of INa(R) with or without the addition of SSM was constructed. The presence of SSM (1 μM) was capable of decreasing INa(R) with no noticeable change in its voltage dependence in GH3 cells, since the overall shape of the I-V curves for INa(R) appearing between the absence and presence of SSM appeared to be similar. For example, at the level of −30 mV, the exposure to 1 μM SSM resulted in a decrease in INa(R) amplitude from 46 ± 6 to 22 ± 5 pA (n = 8, P < 0.05). . Data analyses were done by ANOVA-2 for repeated measures, P(factor 1) < 0.05, P(factor 2) < 0.05, P(interaction) < 0.05, followed by Duncan's post hoc test, P < 0.05. * Significantly different from controls measured at the same level of membrane potential (P < 0.05).

Concentration-Dependent Inhibition of M-Type K + Currents (IK(M)) Caused by SSM in GH3 Cells
The following experiments were further undertaken to determine whether the addition of SSM could exert any perturbations on the amplitude or gating of IK(M) in these cells [34][35][36]. Cells were bathed in high-K + , Ca 2+ -free solution, and the recording pipette was then filled with K + -enriched solution. Figure 5 illustrates that the presence of SSM can result in a concentration-dependent depression in the amplitude of IK(M) during step depolarization. The IC50 value needed for an . Inhibitory effect of SSM on resurgent I Na (I Na(R) ) recorded from GH 3 cells. (A) Representative I Na(R) achieved when the examined cell was depolarized from −80 to +20 mV for 40 msec, then repolarized to various potentials ranging between −50 and 0 mV. a: control; b: 1 µM SSM. (B) Average I-V relationship of I Na(R) attained in the control ( ) or during cell exposure to 1 µM SSM ( ) (mean ± SEM; n = 8 for each point). Of note, the overall I-V relationship of I Na(R) remains unaltered in the presence of SSM, though this agent is able to decrease the amplitude of I Na(R) . Data analyses were done by ANOVA-2 for repeated measures, P(factor 1) < 0.05, P(factor 2) < 0.05, P(interaction) < 0.05, followed by Duncan's post hoc test, P < 0.05. * Significantly different from controls measured at the same level of membrane potential (P < 0.05).

Concentration-Dependent Inhibition of M-Type K + Currents (I K(M) ) Caused by SSM in GH 3 Cells
The following experiments were further undertaken to determine whether the addition of SSM could exert any perturbations on the amplitude or gating of I K(M) in these cells [34][35][36]. Cells were bathed in high-K + , Ca 2+ -free solution, and the recording pipette was then filled with K + -enriched solution. Figure 5 illustrates that the presence of SSM can result in a concentration-dependent depression in the amplitude of I K(M) during step depolarization. The IC 50 value needed for an SSM-perturbed decrease of I K(M) observed in GH 3 cells was 4.8 µM, a value noticeably higher than that used for its inhibitory effect on the late component of I Na in response to brief depolarization.

Inhibitory Effect of SSM on Erg-Mediated K + Current (IK(erg)) in GH3 Cells
We further explored whether SSM could perturb another types of K + currents (i.e., IK(erg) and IK(DR)) in these cells. As demonstrated previously [15,23,37], to amplify the deactivated IK(erg), cells were bathed in high-K + , Ca 2+ -free solution. In this stage of the measurements, we bathed cells in high-K + , Ca 2+ -free solution containing 1 μM tetrodotoxin (TTX), and then filled up the electrodes by using K + -enriched solution. As depicted in Figure 6A,B, as cells were exposed to 30 μM SSM, the amplitude of IK(erg) in response to negative potentials from −10 mV was evidently decreased. Figure  6B represents the average I-V relationship of deactivated IK(erg) achieved in controls and during the exposure to 30 μM SSM. Therefore, SSM at a concentration higher than 30 μM can effectively depress the amplitude of IK(erg) in GH3 cells.

Inhibitory Effect of SSM on Erg-Mediated K + Current (I K(erg) ) in GH 3 Cells
We further explored whether SSM could perturb another types of K + currents (i.e., I K(erg) and I K(DR) ) in these cells. As demonstrated previously [15,23,37], to amplify the deactivated I K(erg) , cells were bathed in high-K + , Ca 2+ -free solution. In this stage of the measurements, we bathed cells in high-K + , Ca 2+ -free solution containing 1 µM tetrodotoxin (TTX), and then filled up the electrodes by using K + -enriched solution. As depicted in Figure 6A,B, as cells were exposed to 30 µM SSM, the amplitude of I K(erg) in response to negative potentials from −10 mV was evidently decreased. Figure 6B represents the average I-V relationship of deactivated I K(erg) achieved in controls and during the exposure to 30 µM SSM. Therefore, SSM at a concentration higher than 30 µM can effectively depress the amplitude of I K(erg) in GH 3

Mild Inhibitory Effect of SSM on Delayed Rectifier K + Currents (IK(DR)) in GH3 Cells
We next examined whether the presence of SSM is able to modify IK(DR) present in GH3 cells. To achieve this goal, we bathed cells in Ca 2+ -free Tyrode's solution which contained 1 μM TTX, and then filled up the recording electrode by using K + -containing solution. When cells were exposed to SSM at a concentration of 3 μM, the amplitude of IK(DR) in response to a 1-sec step depolarization was unchanged. It is noted, however, that the IK(DR) amplitudes responding to different levels of depolarizing command steps were decreased by the addition of 10 μM SSM, though the activation time course of IK(DR) evoked by membrane depolarization remained unchanged. For example, as the IK(DR) amplitude was measured at the level of +40 mV, the presence of 10 μM SSM significantly decreased the current amplitude by 23 ± 4% from 598 ± 34 to 454 ± 29 pA (n = 8, P < 0.05). The average I-V relationship of IK(DR) obtained in the absence or presence of 10 μM was constructed and is hence illustrated in Figure 7. Each I K(erg) amplitude was measured at the beginning of a 1-sec hyperpolarization from −10 mV to various voltages ranging between −100 and −10 mV in 10-mV increments. Data analyses were done by ANOVA-2 for repeated measures, P(factor 1) < 0.05, P(factor 2) < 0.05, P(interaction) < 0.05, followed by Duncan's post hoc test, P < 0.05. * Significantly different from controls measured at the same level of membrane potential (P < 0.05).

Mild Inhibitory Effect of SSM on Delayed Rectifier K + Currents (I K(DR) ) in GH 3 Cells
We next examined whether the presence of SSM is able to modify I K(DR) present in GH 3 cells. To achieve this goal, we bathed cells in Ca 2+ -free Tyrode's solution which contained 1 µM TTX, and then filled up the recording electrode by using K + -containing solution. When cells were exposed to SSM at a concentration of 3 µM, the amplitude of I K(DR) in response to a 1-sec step depolarization was unchanged. It is noted, however, that the I K(DR) amplitudes responding to different levels of depolarizing command steps were decreased by the addition of 10 µM SSM, though the activation time course of I K(DR) evoked by membrane depolarization remained unchanged. For example, as the I K(DR) amplitude was measured at the level of +40 mV, the presence of 10 µM SSM significantly decreased the current amplitude by 23 ± 4% from 598 ± 34 to 454 ± 29 pA (n = 8, P < 0.05). The average I-V relationship of I K(DR) obtained in the absence or presence of 10 µM was constructed and is hence illustrated in Figure 7.

Inability of SSM to Perturb Hyperpolarization-Activated Cation Currents (Ih) in GH3 Cells
In the following experiments, we further studied whether the presence of SSM could perturb another type of inwardly directed current, i.e., Ih. Cells were exposed to Ca 2+ -free Tyrode's solution containing 1 μM TTX and the pipette was filled with K + -containing solution. As the hyperpolarizing command pulse from −40 to −110 mV with a duration of 2 sec was delivered, Ih with a slowly activating property was robustly evoked, as observed previously [16,38,39]. As illustrated in Figure  8, 1 min of exposure to SSM at a concentration of 10 μM was unable to modify the amplitude or gating (i.e., activation or deactivation kinetics) of Ih in response to a 2-sec hyperpolarizing pulse from −40 to −110 mV. For example, at the level of −110 mV, the Ih amplitude measured at the end of the hyperpolarizing step between the absence and presence of 10 μM SSM did not differ (388 ± 24 pA (control) versus 386 ± 26 pA (in the presence of SSM); n = 9, P > 0.05). Similarly, the application of 10 μM SesA had a minimal effect on Ih amplitude. However, in the continued presence of SSM (10 μM), the subsequent application of cilobradine at a concentration of 3 or 10 μM was highly effective at inhibiting the Ih amplitude in combination with a measurable slowing in the activation time course of the current. Cilobradine has recently been reported to decrease Ih amplitude, as well as to alter activation kinetics present in different types of excitable cells [39]. As such, distinguishable from its effect on INa or different types of K + currents demonstrated above, the addition of SSM failed to alter the amplitude and kinetics of Ih identified in GH3 cells.

Inability of SSM to Perturb Hyperpolarization-Activated Cation Currents (I h ) in GH 3 Cells
In the following experiments, we further studied whether the presence of SSM could perturb another type of inwardly directed current, i.e., I h . Cells were exposed to Ca 2+ -free Tyrode's solution containing 1 µM TTX and the pipette was filled with K + -containing solution. As the hyperpolarizing command pulse from −40 to −110 mV with a duration of 2 sec was delivered, I h with a slowly activating property was robustly evoked, as observed previously [16,38,39]. As illustrated in Figure 8, 1 min of exposure to SSM at a concentration of 10 µM was unable to modify the amplitude or gating (i.e., activation or deactivation kinetics) of I h in response to a 2-sec hyperpolarizing pulse from −40 to −110 mV. For example, at the level of −110 mV, the I h amplitude measured at the end of the hyperpolarizing step between the absence and presence of 10 µM SSM did not differ (388 ± 24 pA (control) versus 386 ± 26 pA (in the presence of SSM); n = 9, P > 0.05). Similarly, the application of 10 µM SesA had a minimal effect on I h amplitude. However, in the continued presence of SSM (10 µM), the subsequent application of cilobradine at a concentration of 3 or 10 µM was highly effective at inhibiting the I h amplitude in combination with a measurable slowing in the activation time course of the current. Cilobradine has recently been reported to decrease I h amplitude, as well as to alter activation kinetics present in different types of excitable cells [39]. As such, distinguishable from its effect on I Na or different types of K + currents demonstrated above, the addition of SSM failed to alter the amplitude and kinetics of I h identified in GH 3 cells.

Simulations of SSM-Mediated Inhibition of INa Derived From a Markov State Model
To further elucidate the ionic mechanism of the inhibitory actions of SSM, a modified Markovian model used to simulate INa (i.e., SCN8A-encoded (or NaV1.6) current) was examined. The mRNA transcripts for the α-subunit of NaV1.1, NaV1.2, NaV1.3, and NaV1.6 were reported to be present in GH3 cells [40,41]. This model, illustrated in Figure 9A, was originally derived from Pan and Cummins [21]. The detailed meanings for the default parameters used in this model were previously elaborated [21]. Basically, the model consists of five closed states, one open state, one blocked state, and six inactivation states. As shown in Figure 9B, the inhibitory effect of SSM on simulated INa closely resembled the experimental observations reported above. The observations showed that the inhibitory effect of SSM at a concentration of 0.3 and 1 μM can be mimicked by an increase in Oon (i.e., transitional rate from the open to I6 state) to 3.5 and 4.6 msec −1 from a control value of 2.3 msec −1 . Therefore, a progression toward the activated state became considerably raised in the presence of 0.3 or 1 μM SSM by 25% or 50%, respectively. Overall, the simulation results produced a good match to the experimental observations which disclosed that, during cell exposure to SSM (0.3 or 1 μM), the current amplitude of simulated INa (i.e., SCN8A-encoded current) in response to a brief depolarization was decreased, along with a reduction in the inactivation time constant. Additionally, on the basis of our analysis, as demonstrated in Figure 9C, when the cells were exposed to SSM, the state probability in the OB state of the channel appeared to be sensitive to a decrease to a greater extent than that in the O state. For example, as the modeled cell was exposed

Simulations of SSM-Mediated Inhibition of I Na Derived From a Markov State Model
To further elucidate the ionic mechanism of the inhibitory actions of SSM, a modified Markovian model used to simulate I Na (i.e., SCN8A-encoded (or Na V 1.6) current) was examined. The mRNA transcripts for the α-subunit of Na V 1.1, Na V 1.2, Na V 1.3, and Na V 1.6 were reported to be present in GH 3 cells [40,41]. This model, illustrated in Figure 9A, was originally derived from Pan and Cummins [21]. The detailed meanings for the default parameters used in this model were previously elaborated [21]. Basically, the model consists of five closed states, one open state, one blocked state, and six inactivation states. As shown in Figure 9B, the inhibitory effect of SSM on simulated I Na closely resembled the experimental observations reported above. The observations showed that the inhibitory effect of SSM at a concentration of 0.3 and 1 µM can be mimicked by an increase in Oon (i.e., transitional rate from the open to I6 state) to 3.5 and 4.6 msec −1 from a control value of 2.3 msec −1 . Therefore, a progression toward the activated state became considerably raised in the presence of 0.3 or 1 µM SSM by 25% or 50%, respectively. Overall, the simulation results produced a good match to the experimental observations which disclosed that, during cell exposure to SSM (0.3 or 1 µM), the current amplitude of simulated I Na (i.e., SCN8A-encoded current) in response to a brief depolarization was decreased, along with a reduction in the inactivation time constant. Additionally, on the basis of our analysis, as demonstrated in Figure 9C, when the cells were exposed to SSM, the state probability in the OB state of the channel appeared to be sensitive to a decrease to a greater extent than that in the O state. For example, as the modeled cell was exposed to 1 µM SSM, the occupancy probability in the O state mildly decreased from 0.57 to 0.52, while that in the OB state resulted in a reduction from 0.079 to 0.046. to 1 μM SSM, the occupancy probability in the O state mildly decreased from 0.57 to 0.52, while that in the OB state resulted in a reduction from 0.079 to 0.046. The state diagram of a Markovian model for the NaV channel (i.e., SCN8A channel) depicted in (A) was adopted from a recent study [42]. The solutions to the ordinary differential equations in the current study were implemented in the XPP software package, and the default values for detailed numerical parameters are identical to those demonstrated previously [42], except that the values of ε (i.e., voltage-independent transition rate from the open to blocked state) and maximal conductance of INa in the control (i.e., SSM was not present) were arbitrarily assigned to be 0.3 msec −1 and 3.6 nS, respectively. In (A), C: closed state; O: open state; OB: blocked state; I: inactivated state. Oon and Ooff represent the on and off transition rates for the normal inactivation of NaV channels from the open (O) state, respectively, and are independent of voltage, while Con and Coff are the on and off rates for normal inactivation from the C1-C5 states and is also independent of voltage. a = (Oon/Con) 1/4 and b = (Ooff/Coff) 1/4 . In (B), the model cell was abruptly depolarized from −80 to −10 mV, then repolarized to -50 mV (as indicated in the inset). When the value of Oon was raised to 3.  The state diagram of a Markovian model for the Na V channel (i.e., SCN8A channel) depicted in (A) was adopted from a recent study [42]. The solutions to the ordinary differential equations in the current study were implemented in the XPP software package, and the default values for detailed numerical parameters are identical to those demonstrated previously [42], except that the values of ε (i.e., voltage-independent transition rate from the open to blocked state) and maximal conductance of I Na in the control (i.e., SSM was not present) were arbitrarily assigned to be 0.3 msec −1 and 3.6 nS, respectively. In (

Discussion
The principal findings obtained in the present study are as follows. First, in pituitary GH 3 cells, SSM or SesA, known to be the therapeutic furofuran lignans of sesame oil [1,3,14], differentially and effectively inhibited the transient and late components of I Na in a concentration-dependent manner. Second, the addition of SSM can result in a modification of the inactivation kinetics of I Na in response to brief depolarization. Third, the presence of SSM could inhibit the amplitude of I Na(R) . Fourth, its presence concentration-dependently depressed the amplitude of I K(M) . Fifth, the presence of SSM mildly decreased the amplitude of I K(erg) and I K(M) . Sixth, SSM itself was unable to alter the amplitude or gating of hyperpolarization-elicited I h . Seventh, according to a Markovian model designed from the SCN8A channel adopted previously [21], SSM-perturbed changes in the gating kinetics of Na V channels could be predictably described from their lowering of the probability of open (O) and open-blocked (OB) states of the channel. Overall, the experimental and simulation results found here meant that the inhibition by SSM of these ion channels can be caused by one of several ionic mechanisms underlying its remarkable changes to the functional activities of different types of electrically excitable cells, supposing that similar observations can be found in vivo. To what extent these compounds have therapeutic relevance in the treatment of patients with epilepsy remains to be studied.
A noticeable feature of the block of I Na caused by SSM in GH 3 cells is that the initial rising phase of the current (i.e., activation time course) was unaffected. However, the inhibitory effects of SSM on I Na are not restricted to its suppression of the peak component of the current. As was expected, increasing the SSM concentration not only decreased the peak component of I Na responding to rapid membrane depolarization, but also accelerated the inactivation rate of the current. The SSM molecule appeared in the blocking only when the Na V channel was in the open state. This feature can be incorporated into a simple kinetic scheme (i.e., closed ↔ open ↔ open-blocked), as demonstrated in Figure 1C. As such, it is most likely that SSM or SesA preferentially binds to and blocks the open state of the Na V channels.
In this study, we observed that SSM at the concentrations falling in the range between 0.1 and 0.3 µM caused little or no effect on the peak component of I Na in response to brief membrane depolarization, whereas, at the same concentration, it effectively blocked the sustained component of I Na . In this scenario, the calculated IC 50 value of SSM, which was required for the inhibition of sustained I Na , tends to be lower than that for its inhibitory effect on peak I Na , highly reflecting that there is a considerable and selective block of sustained I Na caused by SSM. Meanwhile, the exposure to SSM produced a reduction in the amplitude of I Na(R) , though no change in the overall I-V relationship of I Na(R) was obtained in its presence.
Sesame oil was shown to exert protective effects against cypermethrin-induced damage in genomic DNA and histopathological changes in the brain or hematotoxicities [13,43]. It was reported to prevent the deleterious effect of cypermethrin in rat liver and kidney [44,45]. The present observations showed that the SSM-mediated inhibition of I Na could be counteracted by a further application of tefluthrin, structurally similar to cypermethrin, suggesting that pyrethroid-induced neurotoxicity could be reversed by SSM or SesA.
It should be noticed that the neurological or cardioprotective actions caused by SSM, SesA, or other structurally similar compounds, as described previously [42,[46][47][48][49][50][51][52][53], can be intimately linked to their direct actions on the amplitude and gating of ion currents (e.g., I Na ). Similar to the ranolazine or perampanel action on I Na described previously [23,25], the inhibitory effect of SSM on ion currents seen herein may be responsible for its wide spectrum of effects observed in vivo [3,54]. Additionally, caution needs to be taken in the interpretation of sesame oil as a fat-soluble vehicle [55][56][57].
The present observations also revealed that SSM could decrease the amplitude of I K(M) in GH 3 cells with an IC 50 of 4.8 µM. I K(M) is biophysically characterized by a slow activation and deactivation property during step depolarization [34][35][36]. It needs to be noticed, therefore, that the inhibition of I Na caused by SSM or SesA could be indirectly and concurrently altered by their inhibitory effects on I K(M) observed in non-voltage-clamped cells, since the suppression of I Na amplitude would be further exacerbated by the membrane depolarization produced by I K(M) inhibition. In other words, the SSM-mediated inhibition of I Na and I K(M) studied herein likely synergistically influences the functional activities of electrically excitable cells such as pituitary lactotrophs. However, whether different lignans in dietary vegetables produce similar actions to the ones observed here still remains to be further examined.
The voltage-clamp current measurements are unable to realize the changes of the occupancy probability of each state simultaneously. In this study, the biophysical model ( Figure 9A) adopted in the present study [21] tends to be based on a relatively small number of variables. However, it allowed us to virtually highlight a qualitative way of how the presence of SSM perturbs the amplitude and gating of I Na . As such, the model demonstrated herein is able to complement the experimental observations by providing insight into the gating of Na V channels, which can impinge upon the electrical behavior of neurons or neuroendocrine cells. Our simulation results generated from this model support the notion that changes in the magnitude and kinetics of I Na caused by SSM, in which varying value of Oon is the valuable parameter involved, are responsible for its actions on the functional activity of electrically excitable cells in vivo, though other additional variables also likely take part in the regulation of I Na kinetics. Oon, appearing in the model, is the on rate of normal inactivation from the open state of the Na V channel. Overall, the findings from the present simulations disclose that the decreases in both peak amplitude and the inactivation time constant of I Na , in which the SSM action is mimicked, could be a potentially important mechanism underlying the rate and pattern of repetitive firing in electrically excitable cells appearing in vivo.

Electrophysiological Measurements
On the day of each experiment, cells were dispersed with a 1% trypsin/EDTA solution and a few drops of cell suspension were rapidly placed in a custom-built recording chamber mounted on the stage of an inverted DM-IL microscope (Leica; Major Instruments, Kaohsiung, Taiwan). They were immersed at room temperature (20-25 • C) in normal Tyrode's solution, the composition of which is elaborated above. We measured ion currents in the whole-cell model of a standard patch-clamp technique with dynamic adaptive suctioning (i.e., decremental change of suction pressure in response to a progressive increase in the electrode resistance), with the aid of an RK-400 (Bio-Logic, Claix, France) or an Axopatch-200B (Molecular Devices, Sunnyvale, CA) patch amplifier [33,38,58]. The microelectrodes used were prepared from Kimax-51 borosilicate capillaries with a 1.5-mm outer diameter (#34500; Kimble; Dogger, New Taipei City, Taiwan) by using a PP-830 vertical puller (Narishige, Taiwan Instrument, Taipei, Taiwan). The recording electrodes had their tip resistances, which ranged between 3 and 5 MΩ, as they were filled up with the different internal solutions elaborated above. During the measurements, the recorded area on the vibration-free table was shielded by using a Faraday cage (Scitech, Seoul, South Korea). The potentials were corrected for the liquid-liquid junction potential that would appear when the composition of the pipette solution remained different from that in the bath.

Data Recordings
The signals, composed of potential and current traces, were monitored on an HM-507 oscilloscope (Hameg, East Meadow, NY) and digitally stored online at 10 kHz in a Sony VAIO CS series laptop computer (VGN-CS110E; Kaohsiung, Taiwan) equipped with a 12-bit resolution Digidata 1440A interface (Molecular Devices). During the recordings with either analog-to-digital or digital-to-analog conversion, the latter device was controlled by pCLAMP 10.7 software (Molecular Devices) run on Microsoft Windows 10 (Redmond, WA). The laptop computer used was also put on the top of an adjustable Cookskin stand (Ningbo, Zheijiang, China) for efficient manipulation during the experiments.

Data Analyses
The digitized signals were examined and analyzed offline using different programs, such as pCLAMP 10.7 (Molecular Devices), 64-big OriginPro 2016 (OriginLab, Taipei, Taiwan), Prism 6 (GraphPad; SoftHome International, Taipei, Taiwan), or custom-made macros created in Microsoft Excel ® 2013, which was executed on Windows 10 (Redmond, WA). The concentration-response data for the inhibition of either peak and late I Na and I K(M) inherently in GH 3 cells were least-squares fitted to the modified Hill equation, which can be written as follows: where [C] denotes the SSM or SesA concentration given; IC 50 and n H represent the concentration required for a 50% inhibition and the Hill coefficient, respectively; and E max is the maximal inhibition of either peak and late I Na or I K(M) caused by the different concentrations of SSM or SesA.

Statistical Analyses
Linearized or non-linearized curve fitting to the data sets was performed using either pCLAMP 10.7 (Molecule Devices), OriginPro (OriginLab), or Prism 6.0 (GraphPad). All data are presented as mean value ± SEM with sample sizes (n) indicative of the cell numbers from which the data were collected; error bars are plotted as SEM. Paired or unpaired Student's t-tests were initially applied for the statistical analyses. As the statistical difference among different groups was necessarily determined, we performed either analysis of variance (ANOVA)-1 or ANOVA-2 with or without repeated measures followed by Duncan's post hoc test. A P-value of < 0.05 was considered to indicate statistical difference.

Computer Simulations
To simulate both the increase in the degree of the I Na inactivation rate and the decrease in the peak I Na , a modified Pan-Cummins model was mathematically constructed in the study. The state model of the SCN8A-encoded (or Na V 1.6) channel which we employed in this work has been described in previous studies [21,59,60]. Such a kinetic scheme that can take into account the obtained results is described below, where C is the final closed state before opening, O is an open state, I is an inactivated state, and OB is a blocked state. The simple Scheme 1 is given as follows: Molecules 2020, 25, x FOR PEER REVIEW 16 of 20

Statistical Analyses
Linearized or non-linearized curve fitting to the data sets was performed using either pCLAMP 10.7 (Molecule Devices), OriginPro (OriginLab), or Prism 6.0 (GraphPad). All data are presented as mean value ± SEM with sample sizes (n) indicative of the cell numbers from which the data were collected; error bars are plotted as SEM. Paired or unpaired Student's t-tests were initially applied for the statistical analyses. As the statistical difference among different groups was necessarily determined, we performed either analysis of variance (ANOVA)-1 or ANOVA-2 with or without repeated measures followed by Duncan's post hoc test. A P-value of < 0.05 was considered to indicate statistical difference.

Computer Simulations
To simulate both the increase in the degree of the INa inactivation rate and the decrease in the peak INa, a modified Pan-Cummins model was mathematically constructed in the study. The state model of the SCN8A-encoded (or NaV1.6) channel which we employed in this work has been described in previous studies [21,59,60]. Such a kinetic scheme that can take into account the obtained results is described below, where C is the final closed state before opening, O is an open state, I is an inactivated state, and OB is a blocked state. The simple Scheme 1 is given as follows: The programs designed in the present study were written in the XPP simulation package available in http://www.math.pitt.edu/~bard/xpp/xpp.html. Differential equations were solved by a fourth order Runge-Kutta algorithm. Parts of the numerical simulations were also verified with Microsoft Excel [20,61].