Activation of Voltage-Gated Na+ Current by GV-58, a Known Activator of CaV Channels

GV-58 ((2R)-2-[(6-{[(5-methylthiophen-2-yl)methyl]amino}-9-propyl-9H-purin-2-yl)amino]butan-1-ol) is recognized to be an activator of N- and P/Q-type Ca2+ currents. However, its modulatory actions on other types of ionic currents in electrically excitable cells remain largely unanswered. This study was undertaken to explore the possible modifications caused by GV-58 in ionic currents (e.g., voltage-gated Na+ current [INa], A-type K+ current [IK(A)], and erg-mediated K+ current [IK(erg)]) identified from pituitary GH3 lactotrophs. GH3 cell exposure to GV-58 enhanced the transient and late components of INa with varying potencies; consequently, the EC50 values of GV-58 required for its differential increase in peak and late INa in GH3 cells were estimated to be 8.9 and 2.6 μM, respectively. The INa in response to brief depolarizing pulse was respectively stimulated or suppressed by GV-58 or tetrodotoxin, but it failed to be altered by ω-conotoxin MVIID. Cell exposure to this compound increased the recovery of INa inactivation evoked by two-pulse protocol based on a geometrics progression; however, in its presence, there was a slowing in the inactivation rate of current decay evoked by a train of depolarizing pulses. The existence of GV-58 also resulted in an increase in the amplitude of ramp-induced resurgent and window INa. The presence of this compound inhibited IK(A) magnitude, accompanied by a shortening in inactivation time course of the current; however, it mildly decreased IK(erg). Under current-clamp conditions, GV-58 increased the frequency of spontaneous action potentials in GH3 cells. Moreover, in NSC-34 motor neuron-like cells, the presence of GV-58 not only raised INa amplitude but also reduced current inactivation. Taken together, the overall work provides a noticeable yet unidentified finding which implies that, in addition to its agonistic effect on Ca2+ currents, GV-58 may concertedly modify the amplitude and gating kinetics of INa in electrically excitable cells, hence modifiying functional activities in these cells.

It has been established that nine isoforms (i.e., Na V 1.1-1.9 [or SCN1A-SCN5A and SCN8A-SCN11A]) of voltage-gated Na + (Na V ) channels are expressed in mammalian excitable tissues, which include central or peripheral nervous system and endocrine system [10,11]. The imperative role played by the activity of these channels is to depolarize the membrane and generate the upstroke of the action potential (AP), hence controlling the firing amplitude, frequency, and pattern present in excitable cells [10][11][12]. Of additional interest, several activators of Na V channels have been elaborated preferentially to slow the inactivation rate as well as to increase the late component of voltage-gated Na + current (I Na ), such as tefluthrin, telmisartan, and apocynin [13][14][15], whereas some Na V channel inhibitors (e.g., ranolazine and esaxerenone) known to increase the inactivation rate of the current have been demonstrated [16,17]. However, at present, whether the existence of GV-58 is capable of interacting with the Na V channels to modify the magnitude and gating properties of I Na in excitable cells remains not very thoroughly studied, although its effectiveness in stimulating N-and P/Q-type Ca 2+ currents has been demonstrated [1][2][3][4][5].
In light of the above-mentioned considerations, in this study, the electrophysiological effects of GV-58 and other related compounds in pituitary GH 3 cells were extensively investigated. We sought to (a) evaluate whether the presence of GV-58 causes any modifications in the amplitude and gating of I Na intrinsically in GH 3 cells; (b) compare the effect of this compound on the peak and late components of I Na activated in response to brief depolarizing pulse; (c) study the effect of GV-58 on erg-mediated K + current (I K(erg) ); (d) assess its possible perturbations on spontaneous APs; and (e) investigate the effect of GV-58 on I Na in NSC-34 motor neuron-like cells. Our results provide the evidence to reveal that, notwithstanding its ability to stimulate the activity of voltage-gated Ca 2+ (Ca V ) channels, the differential stimulation by GV-58 of peak and late I Na may participate in its regulation of the electrical behaviors of excitable cells (e.g., GH 3 and NSC-34 cells).
The external solutions and pipette solutions used in this work are illustrated in Table 1.

Electrophysiological Measurements
During the few hours before the experiments, we gently dissociated GH 3 or NSC-34 cells with 1% trypsin-EDTA solution and then placed a few drops of cell suspension into a home-made chamber fixed on the working stage of an inverted DM-II fluorescence microscope (Leica; Major Instruments, Tainan, Taiwan). Cells were immersed at room temperature (20-25 • C) in normal Tyrode's solution, the composition of which was detailed in Table 1, and they were allowed to attach to the chamber's bottom. The electrodes that we used to measure were fabricated from Kimax-51 glass tubing with 1.5 mm outer diameter (#34500; Kimble, Dogger, New Taipei City, Taiwan) by using a vertical two-stage puller (PP-830; Narishige, Major Instruments, Tainan, Taiwan). Being filled with different internal solution, the recording electrodes had tip resistances of 3-5 MΩ. During the measurements, the electrode was mounted in an air-tight holder which has a suction port on the side, and a chloride silver was used to be in contact with the internal solution. We recorded ionic currents or membrane potential in the whole-cell configuration of a modified patch-clamp technique with the use of either an Axoclamp-2B (Molecular Devices, Sunnyvale, CA, USA) or an RK-400 amplifier (Bio-Logic, Claix, France), as elaborated elsewhere [13][14][15]. GΩ-seals were commonly achieved in an all-or-nothing fashion and resulted in a dramatic improvement in signal-to-noise ratio. The liquid junction potentials (−13 ± 1 mV, n = 27), which occur when the ionic compositions in the pipette internal solution and those of bath solution are different, were zeroed shortly before GΩ-seal formation was achieved, and the whole-cell data were then corrected. During measurements, we exchanged the solutions through a home-made gravity-driven type of bath perfusion.
The signals (i.e., current and potential tracings) were monitored at a given interval and digitally stored on-line at 10 kHz in an ASUS ExpertBook laptop computer (P2451F; Yuan-Dai, Tainan, Taiwan). For efficient analog-to-digital (A/D) and digital-to-analog (D/A) conversion to proceed, a Digidata-1440A equipped with a laptop computer was operated by pClamp 10.6 software run under Microsoft Windows 7 (Redmond, WA, USA).
We low-pass filtered current signals at 2 kHz with a FL-4 four-pole Bessel filter (Dagan, Minneapolis, MN, USA). A variety of pClamp-generated voltage-clamp protocols with various rectangular or ramp waveforms were designed and then delivered to the tested cells through D/A conversion in order to determine the current-voltage (I-V) relation of ionic currents specified (Sections 3.3 and 3.10) and the recovery time course of current inactivation (Section 3.4). As pulse-train stimulation was applied to the tested cell, we used an Astro-Med Grass S88X dual output pulse stimulator (Grass, West Warwick, RI, USA).

Data Analyses
To assess concentration-dependent stimulation of GV-58 on the transient (peak) and late components of I Na , we kept cells bathed in Ca 2+ -free Tyrode's solution. During the recordings, we voltage-clamped each examined cell at a holding potential of −80 mV, and the brief depolarizing pulse to −10 mV was delivered to evoke peak and late I Na . The late I Na during cell exposure to 100 µM GV-58 was taken as 100%, and those (i.e., peak and late I Na ) acquired in the presence of different GV-58 concentrations (0.3-30 µM) were then compared. The concentration-dependent stimulation by this compound of peak and late I Na in pituitary GH 3 cells were determined by fitting the modified Hill equation. That is, where [GV-58] is the GV-58 concentration applied, n H the Hill coefficient, EC 50 the concentration required for a 50% stimulation of peak or late I Na activated in response to rapid depolarizing command voltage, and E max the maximal stimulation of peak or late I Na produced by the existence of GV-58.
The I-V relationship of peak I Na with or without addition of GV-58 was derived and fitted with a Boltzmann function given by: where V is the voltage in mV, E rev the reversal potential of I Na (fixed at +45 mV), G the Na + conductance in nS, and I the current in pA, while V h and k are the gating parameters.

Curve-Fitting Procedures and Statistical Analyses
Curve-fitting (linear or non-linear [e.g., exponential or sigmoidal curve]) to experimental datasets was conducted with the goodness of fit by using different analytical procedures, such as the Microsoft "Solver" built in Excel 2019 (Microsoft), OriginPro ® 2021 program (OriginLab; Scientific Formosa, Kaohsiung, Taiwan), and MathCad Prime 7 (Otsuka, New Taipei City, Taiwan). The electrophysiological data are presented as the mean ± standard error of the mean (SEM), with the sample sizes (n) indicating the cell numbers from which the results were acquired. We performed Student's t-tests (for paired or unpaired samples) and analysis of variance (one-way ANOVA or two-way-ANOVA) with or without repeated measures followed by post hoc Fisher's least significance difference test. The statistical calculations were performed by using SPSS 17.0 (AsiaAnalytics, Taipei, Taiwan). A p-value of less than 0.05 was considered to indicate statistical difference.

Stimulatory Effect of GV-58 on Voltage-Gated Na + Current Measured from Pituitary GH 3 Cells
For the first stage of measurements, we kept cells bathed in Ca 2+ -free Tyrode's solution which contained 10 mM tetraethylammonium chloride (TEA) and 0.5 mM CdCl 2 . TEA and CdCl 2 were used to block most of the K + and Ca 2+ currents, respectively. As the depolarizing command voltage from −80 to −10 mV was imposed on the tested cells, we were able to detect an inward current which displayed the rapidly activating and inactivating time course ( Figure 1A,B). This type of inward current was, respectively, sensitive to inhibition or stimulation by tetrodotoxin (1 µM) or tefluthrin (10 µM), and it has hence been identified as a voltage-gated Na + current (I Na ) [13,17,19]. Of notice, one minute after cells were exposed to GV-58, the amplitude of I Na progressively increased in combination with a slowing in the inactivation time course of the current. For example, the addition of GV-58 markedly increased the peak I Na from 215 ± 21 to 309 ± 29 pA (n = 8, p < 0.05), and the time constant in the slow component of current inactivation (τ inact(S) ) was concurrently raised to 3.8 ± 0.4 ms from a control of 2.1 ± 0.3 ms (n = 8, p < 0.05). However, no significant difference in the fast component of current inactivation could be demonstrated. After washout of the compound, current amplitude was returned to 219 ± 23 pA (n = 7, p < 0.05).
vating time course ( Figure 1A,B). This type of inward current was, respectively, sensitive to inhibition or stimulation by tetrodotoxin (1 μM) or tefluthrin (10 μM), and it has hence been identified as a voltage-gated Na + current (INa) [13,17,19]. Of notice, one minute after cells were exposed to GV-58, the amplitude of INa progressively increased in combination with a slowing in the inactivation time course of the current. For example, the addition of GV-58 markedly increased the peak INa from 215 ± 21 to 309 ± 29 pA (n = 8, p < 0.05), and the time constant in the slow component of current inactivation (τinact(S)) was concurrently raised to 3.8 ± 0.4 ms from a control of 2.1 ± 0.3 ms (n = 8, p < 0.05). However, no significant difference in the fast component of current inactivation could be demonstrated. After washout of the compound, current amplitude was returned to 219 ± 23 pA (n = 7, p < 0.05).
The relationship between the GV-58 concentration and the peak or late component of INa evoked in response to abrupt membrane depolarizing was further analyzed and constructed. In this stage of experiments, each cell was rapidly depolarized from −80 to −10 mV, and peak or late INa measured at varying concentrations (0.3-100 μM) of GV-58 were compared. As can be seen in Figure 1C, GH3 cell exposure to GV-58 resulted in a concentration-dependent rise in the amplitude of peak and late INa. The EC50 value for GV-58-stimulated peak or late INa was computed to be 8.9 or 2.6 μM, respectively, and GV-58 at a concentration of 100 μM almost fully increased INa. The present results, therefore, reflect that GV-58 is capable of exercising a stimulatory action on depolarization-activated INa in GH3 cells and that the late component of INa increases to a greater extent than the peak component of the current in its presence.

Comparisons of Effects of GV-58, ω-Conotoxin MVIID, and Tetrodotoxin (TTX) on Peak INa
Earlier studies have been demonstrated to report the effectiveness of GV-58 in enhancing the amplitude of voltage-gated Ca 2+ currents (e.g., N-and P/Q-type Ca 2+ currents) [2][3][4][5]. We, therefore, explored and compared effects of GV-58, ω-conotoxin MVIID, or TTX on INa inherently in these cells. ω-Conotoxin MVIID, a small, disulfide-rich peptide purified from the venoms of predatory cone snails, was previously reported to be an inhibitor of N-and P/Q-type Ca 2+ currents in adrenal chromaffin cells [20]. As summarized in Figure 2, the peak amplitude of INa was increased in the presence of GV-58; however, it was effectively depressed by cell exposure to TTX (1 μM) but not by ω-conotoxin MVIID (100 nM). Moreover, the amplitude of L-type Ca 2+ currents (ICa,L) evoked from −50 to +10 mV . Current trace labeled "a" was obtained during control (i.e., absence of GV-58), those labeled "b" or "c" were recorded during the exposure to 0.3 or 1 µM GV−58, and that labeled "d" was obtained in the presence of 1 µM GV-58 plus 1 µM tetrodotoxin (TTX). In (B), current traces (indicated in open circles) indicate an expanded record from dashed box of (A) for a higher time resolution. Of note, current trace labeled "d" was skipped. The gray lines overlaid on each trace indicate best fit to the data points with a two-exponential function. (C) Concentration−dependent response of GV−58−mediated stimulation of peak or late I Na residing in GH 3 cells (mean ± SEM; n = 8). The peak and late components of I Na activated in response to the depolarizing pulse from −80 to −10 mV were taken at the start and the end−pulse of the depolarizing command voltage with a duration of 80 ms. The continuous line (blue and red colors) indicates the best fit to a modified Hill equation, as revealed in Materials and Methods.
The relationship between the GV-58 concentration and the peak or late component of I Na evoked in response to abrupt membrane depolarizing was further analyzed and constructed. In this stage of experiments, each cell was rapidly depolarized from −80 to −10 mV, and peak or late I Na measured at varying concentrations (0.3-100 µM) of GV-58 were compared. As can be seen in Figure 1C, GH 3 cell exposure to GV-58 resulted in a concentration-dependent rise in the amplitude of peak and late I Na . The EC 50 value for GV-58-stimulated peak or late I Na was computed to be 8.9 or 2.6 µM, respectively, and GV-58 at a concentration of 100 µM almost fully increased I Na . The present results, therefore, reflect that GV-58 is capable of exercising a stimulatory action on depolarization-activated I Na in GH 3 cells and that the late component of I Na increases to a greater extent than the peak component of the current in its presence.

Comparisons of Effects of GV-58, ω-Conotoxin MVIID, and Tetrodotoxin (TTX) on Peak I Na
Earlier studies have been demonstrated to report the effectiveness of GV-58 in enhancing the amplitude of voltage-gated Ca 2+ currents (e.g., N-and P/Q-type Ca 2+ currents) [2][3][4][5]. We, therefore, explored and compared effects of GV-58, ω-conotoxin MVIID, or TTX on I Na inherently in these cells. ω-Conotoxin MVIID, a small, disulfide-rich peptide purified from the venoms of predatory cone snails, was previously reported to be an inhibitor of N-and P/Q-type Ca 2+ currents in adrenal chromaffin cells [20]. As summarized in Figure 2, the peak amplitude of I Na was increased in the presence of GV-58; however, it was effectively depressed by cell exposure to TTX (1 µM) but not by ω-conotoxin MVIID (100 nM). Moreover, the amplitude of L-type Ca 2+ currents (I Ca,L ) evoked from −50 to +10 mV was not affected by ω-conotoxin MVIID (100 nM) or by GV-58 (3 µM). In the continued presence of 3 µM GV-58, further addition of nimodipine (1 µM), an inhibitor of I Ca,L , effectively decreased the peak amplitude of I Ca,L by about 90%. Therefore, the I Na evoked in response to brief depolarizing pulse is sensitive to stimulation by GV-58 and to inhibition by TTX, but it remains unaltered in the presence of ω-conotoxin MVIID, suggesting that the magnitude of N-and P/Q-type Ca 2+ currents is unlikely to be linked to GV-58-stimulated I Na found in GH 3 cells.
Biomedicines 2022, 10, x FOR PEER REVIEW 7 of 21 Figure 2. Comparisons of effects of GV-58, GV-58 plus ω-conotoxin MVIID, and GV-58 plus tetrodotoxin (TTX) on the peak amplitude of INa recorded from GH3 cells. Cells were immersed in Ca 2+free Tyrode's solution containing 10 mM TEA, and the pipettes used were filled with Cs + -containing solution. Current amplitude was taken at the beginning of the brief depolarizing pulse from −80 to −10 mV. Each bar indicates the mean ± SEM (n = 8 for each bar). Of notice, the peak INa in GH3 cells is subject to inhibition by TTX but not by ω-conotoxin MVIID. * Significantly different from control (p < 0.05) and ** significantly different from GV-58 (3 μM) alone group (p < 0.05). Statistical analyses were made by one-way ANOVA among different groups (p < 0.05).

Effect of GV-58 on the Steady-State Current-Voltage (I-V) Relationship of Peak INa
We further examined the steady-state I-V relationships of peak INa obtained with or without the application of GV-58. These voltage-clamp experiments were conducted in cells held at −80 mV, and a series of voltage pulses between −80 and +40 mV was imposed on the tested cells. As shown in Figure 3, the presence of 1 μM GV-58 did not alter the overall I-V relationship of peak INa, although it increased peak amplitude of INa, particu- Comparisons of effects of GV-58, GV-58 plus ω-conotoxin MVIID, and GV-58 plus tetrodotoxin (TTX) on the peak amplitude of I Na recorded from GH 3 cells. Cells were immersed in Ca 2+ -free Tyrode's solution containing 10 mM TEA, and the pipettes used were filled with Cs +containing solution. Current amplitude was taken at the beginning of the brief depolarizing pulse from −80 to −10 mV. Each bar indicates the mean ± SEM (n = 8 for each bar). Of notice, the peak I Na in GH 3 cells is subject to inhibition by TTX but not by ω-conotoxin MVIID. * Significantly different from control (p < 0.05) and ** Significantly different from GV-58 (3 µM) alone group (p < 0.05). Statistical analyses were made by one-way ANOVA among different groups (p < 0.05).

Effect of GV-58 on the Steady-State Current-Voltage (I-V) Relationship of Peak I Na
We further examined the steady-state I-V relationships of peak I Na obtained with or without the application of GV-58. These voltage-clamp experiments were conducted in cells held at −80 mV, and a series of voltage pulses between −80 and +40 mV was imposed on the tested cells. As shown in Figure 3, the presence of 1 µM GV-58 did not alter the overall I-V relationship of peak I Na , although it increased peak amplitude of I Na , particularly at the level of −10 mV. The reversal potential of peak I Na did not differ significantly between the absence and presence of GV-58. The I-V curves obtained in the control period and during exposure to 1 µM GV-58 were fitted with a Boltzmann function as stated in Materials and Methods. In control (i.e., the absence of GV-58), G = 5.5 ± 0.3 nS, V h = −23.9 ± 2.1 mV, k = 14.9 ± 1.9 (n = 8), while in the presence of 1 µM GV-58, G = 7.5 ± 0.4 nS, V h = −23.6 ± 1.9 mV, k = 14.5 ± 1.8 (n = 8). It is, thus, likely that the steady-state activation curve of I Na was not changed during cell exposure to 1 µM GV-58. Figure 2. Comparisons of effects of GV-58, GV-58 plus ω-conotoxin MVIID, and GV-58 plus tetrodotoxin (TTX) on the peak amplitude of INa recorded from GH3 cells. Cells were immersed in Ca 2+free Tyrode's solution containing 10 mM TEA, and the pipettes used were filled with Cs + -containing solution. Current amplitude was taken at the beginning of the brief depolarizing pulse from −80 to −10 mV. Each bar indicates the mean ± SEM (n = 8 for each bar). Of notice, the peak INa in GH3 cells is subject to inhibition by TTX but not by ω-conotoxin MVIID. * Significantly different from control (p < 0.05) and ** significantly different from GV-58 (3 μM) alone group (p < 0.05). Statistical analyses were made by one-way ANOVA among different groups (p < 0.05).

Effect of GV-58 on the Steady-State Current-Voltage (I-V) Relationship of Peak INa
We further examined the steady-state I-V relationships of peak INa obtained with or without the application of GV-58. These voltage-clamp experiments were conducted in cells held at −80 mV, and a series of voltage pulses between −80 and +40 mV was imposed on the tested cells. As shown in Figure 3, the presence of 1 μM GV-58 did not alter the overall I-V relationship of peak INa, although it increased peak amplitude of INa, particularly at the level of −10 mV. The reversal potential of peak INa did not differ significantly between the absence and presence of GV-58. The I-V curves obtained in the control period and during exposure to 1 μM GV-58 were fitted with a Boltzmann function as stated in Materials and Methods. In control (i.e., the absence of GV-58), G = 5.5 ± 0.3 nS, Vh = −23.9 ± 2.1 mV, k = 14.9 ± 1.9 (n = 8), while in the presence of 1 μM GV-58, G = 7.5 ± 0.4 nS, Vh = −23.6 ± 1.9 mV, k = 14.5 ± 1.8 (n = 8). It is, thus, likely that the steady-state activation curve of INa was not changed during cell exposure to 1 μM GV-58.  In these experiments, we voltage−clamped the cells at −80 mV, and various depolarizing pulses from −80 to +40 in 10 mV increments were applied to evoke I Na . Current amplitude at each depolarizing pulse was measured at the beginning of the voltage pulse. Data points shown in filled blue squares are control, and those in open brown circles were obtained in the presence of 1 µM GV−58. The smooth line taken with or without the GV−58 addition was fitted with a Boltzmann function as detailed in Materials and Methods.

Effect of GV-58 on the Recovery from I Na Inactivation Evoked during Varying Interpulse Intervals
We next examined whether the existence of GV-58 produces any effect on the recovery of I Na from inactivation by responding to a two-step voltage protocol. In this protocol, a 30 ms step to −10 mV was applied to the examined cell, and another 30 ms conditioning step to −10 mV with varying interpulse intervals inactivated most of the current. The recovery from current inactivation at the holding potential of −80 mV was hence examined at different times with a geometrics progression, as demonstrated in Figure 4A,B. In the control period (i.e., GV-58 was not present), the peak amplitude of I Na nearly completely recovered from inactivation when the interpulse duration reached 1 s. The time constants of recovery from current inactivation acquired in the absence and presence of 10 µM GV-58 were least squares fitted by a single-exponential function with the values of 5.34 ± 0.07 and 2.11 ± 0.06 ms (n = 8, p < 0.05), respectively. The experimental observations indicate that a conceivable increase in the recovery from inactivation of I Na is seen by adding GV-58.
control period (i.e., GV-58 was not present), the peak amplitude of INa nearly completely recovered from inactivation when the interpulse duration reached 1 s. The time constants of recovery from current inactivation acquired in the absence and presence of 10 μM GV-58 were least squares fitted by a single-exponential function with the values of 5.34 ± 0.07 and 2.11 ± 0.06 ms (n = 8, p < 0.05), respectively. The experimental observations indicate that a conceivable increase in the recovery from inactivation of INa is seen by adding GV-58.

GV-58 Effect on INa Decay Evoked during a Train of Depolarizing Command Voltages in GH3 Cells
Previous work has shown the ability of the train of depolarizing pulses to perturb the peak and persistent INa, which tend to decrease over time in an exponential fashion [21]. We thus made an effort to explore whether GV-58-mediated stimulation of INa can be perturbed during pulse-train stimulation. In this set of whole-cell current measurements, we delivered a 40 Hz train of depolarizing pulses from −80 to −10 mV to the tested cell. As shown in Figure 5A-C, the slow current inactivation evoked by responding to such pulse-

GV-58 Effect on I Na Decay Evoked during a Train of Depolarizing Command Voltages in GH 3 Cells
Previous work has shown the ability of the train of depolarizing pulses to perturb the peak and persistent I Na , which tend to decrease over time in an exponential fashion [21]. We thus made an effort to explore whether GV-58-mediated stimulation of I Na can be perturbed during pulse-train stimulation. In this set of whole-cell current measurements, we delivered a 40 Hz train of depolarizing pulses from −80 to −10 mV to the tested cell. As shown in Figure 5A-C, the slow current inactivation evoked by responding to such pulsetrain stimulation was prominently observed. The mean relationship of peak I Na versus the train duration taken with or without the addition of 3 µM GV-58 was constructed and then analyzed. It is noticed that the presence of GV-58 results in slowing the time course of current inactivation during such a train of depolarizing pulses. For example, the time constant for I Na during a train of command voltages was measurably increased to 189 ± 23 ms (n = 7, p < 0.05) from a control value of 92 ± 12 ms (n = 7). It is clear, therefore, that the existence of GV-58 produces a marked increase in the decaying time constant of I Na during a train of depolarizing pulses.

Use-Dependence of GV-58-Induced Stimulation of Peak INa
This series of whole-cell experiments was conducted to assess the use-dependent property of GV-58-mediated stimulation of peak INa. As shown in Figure 6, the depolarizing pulses from −80 to −10 mV (30 ms in duration) were applied at 0.2 Hz. Under control conditions (i.e., absence of GV-58), when peak INa remained active in Ca 2+ -free Tyrode's solution, the depolarizing pulses were stopped; thereafter, after 25 s of cessation, the depolarizing pulses from −80 to −10 mV at 2 Hz were given to the cells again. The relative

Use-Dependence of GV-58-Induced Stimulation of Peak I Na
This series of whole-cell experiments was conducted to assess the use-dependent property of GV-58-mediated stimulation of peak I Na . As shown in Figure 6, the depolarizing pulses from −80 to −10 mV (30 ms in duration) were applied at 0.2 Hz. Under control conditions (i.e., absence of GV-58), when peak I Na remained active in Ca 2+ -free Tyrode's solution, the depolarizing pulses were stopped; thereafter, after 25 s of cessation, the depolarizing pulses from −80 to −10 mV at 2 Hz were given to the cells again. The relative amplitude of peak I Na with respect to before the cessation of the depolarizing pulse was constructed and noticed to decline in an exponential fashion. It is illustrated in Figure 5 that peak I Na consisted of both tonic and use-dependent components. For example, in the control period, after a 25 s pause, the peak I Na evoked by the first voltage step or during subsequent repetitive stimuli was suppressed by 11 ± 3% or 38 ± 5% (n = 7). As cells were exposed to GV-58 (1 µM), the peak I Na amplitude at the first depolarizing pulse or during subsequent repetitive pulses were significantly suppressed by 8 ± 2% or 30 ± 4%, respectively; and the decline in peak I Na became slowed. The results indicated that GV-58stimulated I Na tends to be composed of two components, namely, tonic and use-dependent components.

Stimulatory Effect of GV-58 on Resurgent INa (INa(R)) Seen in GH3 Cells
The INa(R) has been recently identified in GH3 cells [19,22]. Consistent with previous observations in neurons or endocrine cells [22,23], the biophysical property of this current is that it remains undetectable until the membrane potential is repolarized below 0 mV. In addition to being activated by negative voltage steps (i.e., repolarizing steps) rather than positive voltage steps, INa(R) was noted to activate and decay more slowly than transient INa. The INa(R) is thought to help produce rapid depolarization immediately after an AP and is suited for cells that fire spontaneously at a higher firing rate [23]. For these reasons, we additionally studied whether GV-58 could exert any perturbations on this current. As the whole-cell configuration was securely established, the depolarizing pulse from −80 to +30 mV followed by a 300 ms descending (or repolarizing) ramp command voltage to −80 mV was applied to the examined cell. As depicted in Figure 7A,B, the INa(R) . Of notice, in the presence of GV-58, the peak I Na activated by the first depolarizing step following a pause (around 25 s) had been suppressed to a lesser extent (i.e., tonic stimulation), and, during the repetitive stimuli, the amplitude of peak I Na was further reduced exponentially to a lesser and slower extent (use−dependent stimulation).

Stimulatory Effect of GV-58 on Resurgent I Na (I Na(R) ) Seen in GH 3 Cells
The I Na(R) has been recently identified in GH 3 cells [19,22]. Consistent with previous observations in neurons or endocrine cells [22,23], the biophysical property of this current is that it remains undetectable until the membrane potential is repolarized below 0 mV. In addition to being activated by negative voltage steps (i.e., repolarizing steps) rather than positive voltage steps, I Na(R) was noted to activate and decay more slowly than transient I Na . The I Na(R) is thought to help produce rapid depolarization immediately after an AP and is suited for cells that fire spontaneously at a higher firing rate [23]. For these reasons, we additionally studied whether GV-58 could exert any perturbations on this current. As the whole-cell configuration was securely established, the depolarizing pulse from −80 to +30 mV followed by a 300 ms descending (or repolarizing) ramp command voltage to −80 mV was applied to the examined cell. As depicted in Figure 7A,B, the I Na(R) amplitude activated by this voltage-clamp protocol was evidently increased. For example, the presence of 3 µM GV-58 conceivably increased I Na(R) at −20 mV from 58 ± 3 to 84 ± 7 pA (n = 7, p < 0.05). The further addition of ranolazine (10 µM), but still in the presence of 3 µM GV-58, reduced I Na(R) amplitude at the same level to 62 ± 5 pA (n = 7, p < 0.05). However, the subsequent application of CdCl 2 (0.5 mM) failed to exert any effects on GV-58-stimulated I Na(R) in GH 3 cells. Ranolazine was previously demonstrated to be an inhibitor of late I Na [16,19].

Effect of GV-58 on the Window Component of INa (INa(W)) Recorded from GH3 Cells
The presence of instantaneous INa(W) has been previously demonstrated in different types of electrically excitable cells [12,[24][25][26]. We continued to explore whether GV-58 presence in GH3 cells could modify the magnitude of INa(W) activated in response to rapid ascending ramp command voltage. In an effort to perform these experiments, we voltageclamped the tested cell at −80 mV, and we then applied an ascending ramp from −80 to +20 mV with a duration of 20 ms to evoke INa(W) [26]. As demonstrated in Figure 8A,B, within one minute of exposing cells to GV-58 (1 or 3 μM), the amplitude of INa(W) achieved by the upsloping ramp with a duration of 20 ms was strikingly increased. For example, the existence of 3 μM GV-58 evidently increased the peak component of INa(W) measured at the level of −20 mV from 378 ± 22 to 498 ± 28 pA (n = 7, p < 0.05). After washout of GV-58, current amplitude was returned to 383 ± 24 pA (n = 7). However, it needs to be noticed that INa(W) measured here was evoked by rapid ramp pulse; it would be unlikely to determine the steady-state activation of INa. The summary bar graph shown in Figure 8B reveals that the GV-58 addition is effective in increasing the INa(W) amplitude and that subsequent addition of 1 μM TTX suppressed GV-58-mediated increase of INa(W). The present results also imply that GV-58-mediated accentuation of INa(W) is not mediated through its agonistic effect on CaV channels. , and GV−58 plus CdCl 2 on the amplitude of I Na(R) (mean ± SEM; n = 8 for each bar). Current amplitudes were measured at the level of −20 mV. Of notice, in the continued presence of GV−58, subsequent application of ranolazine, but not of CdCl 2 , effectively suppresses I Na(R) activated by the descending ramp pulse. * Significantly different from control (p < 0.05) and ** significantly different from GV−58 (3 µM) alone group (p < 0.05). Statistical analyses were performed by one−way ANOVA among different groups (p < 0.05).

Effect of GV-58 on the Window Component of I Na (I Na(W) ) Recorded from GH 3 Cells
The presence of instantaneous I Na(W) has been previously demonstrated in different types of electrically excitable cells [12,[24][25][26]. We continued to explore whether GV-58 presence in GH 3 cells could modify the magnitude of I Na(W) activated in response to rapid ascending ramp command voltage. In an effort to perform these experiments, we voltageclamped the tested cell at −80 mV, and we then applied an ascending ramp from −80 to +20 mV with a duration of 20 ms to evoke I Na(W) [26]. As demonstrated in Figure 8A,B, within one minute of exposing cells to GV-58 (1 or 3 µM), the amplitude of I Na(W) achieved by the upsloping ramp with a duration of 20 ms was strikingly increased. For example, the existence of 3 µM GV-58 evidently increased the peak component of I Na(W) measured at the level of −20 mV from 378 ± 22 to 498 ± 28 pA (n = 7, p < 0.05). After washout of GV-58, current amplitude was returned to 383 ± 24 pA (n = 7). However, it needs to be noticed that I Na(W) measured here was evoked by rapid ramp pulse; it would be unlikely to determine the steady-state activation of I Na . The summary bar graph shown in Figure 8B reveals that the GV-58 addition is effective in increasing the I Na(W) amplitude and that subsequent addition of 1 µM TTX suppressed GV-58-mediated increase of I Na(W) . The present results also imply that GV-58-mediated accentuation of I Na(W) is not mediated through its agonistic effect on Ca V channels.

Inhibitory Effect of GV-58 on A-Type K + Current (IK(A)) in GH3 Cells
The suppression of K + currents was shown to be effective in improving synaptic impairment or muscle weakness in Lambert-Eaton myasthenic syndrome [3,27]. In another separate set of whole-cell current recordings, different types of ionic currents (e.g., IK(A)) were also further examined with respect to possible modifications of GV-58 on them. In these experiments, we bathed cells in Ca 2+ -free Tyrode's solution containing 1 μM TTX and 0.5 mM CdCl2, while the recording electrode was filled up with K + -containing solution. The tested cells were voltage-clamped at −80 mV, and the depolarizing command voltage to 0 mV with a duration of 1 s was delivered to evoke a unique type of K + outward current with rapid activation and inactivation, which is referred to as A-type K + current (IK(A)) [28]. One minute after cells were exposed to GV-58 (1 or 3 μM), the amplitude of IK(A) was found to be conceivably decreased; concurrently, the time constant in the slow component of current inactivation (τinact(S)) was reduced ( Figure 9A-C). For example, the addition of 3 μM GV-58 decreased peak IK(A) amplitude from 423 ± 23 to 121± 14 pA (n = 8, p < 0.05), and it concomitantly reduced the τinact(S) value from 56 ± 7 to 12 ± 3 ms (n = 8, p < 0.05). The presence of 5 mM 4-aminopyridine fully suppressed the amplitude of IK(A) in GH3 cells. Results from these observations reflect that, like the action of 4-aminopyridine on IK(A) described previously [28,29], GV-58 per se is capable of decreasing IK(A) and the τinact(S) value of the current found in GH3 cells.

Inhibitory Effect of GV-58 on A-Type K + Current (I K(A) ) in GH 3 Cells
The suppression of K + currents was shown to be effective in improving synaptic impairment or muscle weakness in Lambert-Eaton myasthenic syndrome [3,27]. In another separate set of whole-cell current recordings, different types of ionic currents (e.g., I K(A) ) were also further examined with respect to possible modifications of GV-58 on them. In these experiments, we bathed cells in Ca 2+ -free Tyrode's solution containing 1 µM TTX and 0.5 mM CdCl 2 , while the recording electrode was filled up with K + -containing solution. The tested cells were voltage-clamped at −80 mV, and the depolarizing command voltage to 0 mV with a duration of 1 s was delivered to evoke a unique type of K + outward current with rapid activation and inactivation, which is referred to as A-type K + current (I K(A) ) [28]. One minute after cells were exposed to GV-58 (1 or 3 µM), the amplitude of I K(A) was found to be conceivably decreased; concurrently, the time constant in the slow component of current inactivation (τ inact(S) ) was reduced ( Figure 9A-C). For example, the addition of 3 µM GV-58 decreased peak I K(A) amplitude from 423 ± 23 to 121± 14 pA (n = 8, p < 0.05), and it concomitantly reduced the τ inact(S) value from 56 ± 7 to 12 ± 3 ms (n = 8, p < 0.05). The presence of 5 mM 4-aminopyridine fully suppressed the amplitude of I K(A) in GH 3 cells. Results from these observations reflect that, like the action of 4-aminopyridine on I K(A) described previously [28,29], GV-58 per se is capable of decreasing I K(A) and the τ inact(S) value of the current found in GH 3 cells. , respectively (mean ± SEM; n = 8 for each bar). * Significantly different from controls (p < 0.05). Current amplitude was taken at the start of each depolarizing command voltage from −80 to 0 mV with a duration of 1 s. Of notice, cell exposure to GV−58 is capable of decreasing both peak IK(A) and the τinact(S) value of the current in these cells.

Mild Suppression of Erg-Mediated K + Current (IK(erg)) Produced by GV-58
In another set of experiments, we further examined the effect of GV-58 on IK(erg) in GH3 cells. This set of measurements was conducted in cells bathed in high-K + , Ca 2+ -free solution which contained 1 μM TTX, and the electrode was filled with K + -containing solution. The tested cell was maintained in −10 mV, and different command voltages ranging between −90 and 0 mV with a duration of 1 s were applied to evoke hyperpolarizationevoked IK(erg) [30,31]. During cell exposure to 3 μM GV-58, the amplitude of IK(erg) was noted to remain unaltered. However, as cells were exposed to 10 μM GV-58, the peak and sustained components of IK(erg) measured at the entire voltage-clamp ranges examined were diminished ( Figure 10). For example, as the hyperpolarizing command voltage from −10 to −90 mV with a duration of 1 s was applied to the tested cell, the peak and sustained components of deactivating IK(erg) were significantly reduced to 283 ± 26 and 16 ± 13 pA (n = 8, p < 0.05) from control values of 336 ± 31 and 28 ± 14 pA (n = 8), respectively. After washout of the compound, peak and sustained IK(erg) was returned to 328 ± 29 and 27 ± 13 pA (n = 7). The mean I-V relationships of peak and sustained IK(erg) acquired in the absence or presence of 10 μM GV-58 were constructed and are hence presented in Figure 10B,C, respectively. However, as cells were exposed to E-4031 (10 μM), an inhibitor of IK(erg), the IK(erg) evoked from −10 to −100 mV was fully abolished. Therefore, as compared with its effect on IK(A), the IK(erg) residing in GH3 cells is mildly suppressed by GV-58 presence. shows an expanded record from the dashed box, while the data points (circle symbols) were reduced by 50 for better illustration. The smooth line in the lower part represents best fit to two−exponential function (i.e., fast and slow components in current inactivation). Summary graphs appearing in (B,C) demonstrate inhibitory effects of GV−58 (1 or 3 µM) on peak amplitude and slow component in inactivation time constant (τ inact(S) ) of I K(A) , respectively (mean ± SEM; n = 8 for each bar). * Significantly different from controls (p < 0.05). Current amplitude was taken at the start of each depolarizing command voltage from −80 to 0 mV with a duration of 1 s. Of notice, cell exposure to GV−58 is capable of decreasing both peak I K(A) and the τ inact(S) value of the current in these cells.

Mild Suppression of Erg-Mediated K + Current (I K(erg) ) Produced by GV-58
In another set of experiments, we further examined the effect of GV-58 on I K(erg) in GH 3 cells. This set of measurements was conducted in cells bathed in high-K + , Ca 2+ -free solution which contained 1 µM TTX, and the electrode was filled with K + -containing solution. The tested cell was maintained in −10 mV, and different command voltages ranging between −90 and 0 mV with a duration of 1 s were applied to evoke hyperpolarization-evoked I K(erg) [30,31]. During cell exposure to 3 µM GV-58, the amplitude of I K(erg) was noted to remain unaltered. However, as cells were exposed to 10 µM GV-58, the peak and sustained components of I K(erg) measured at the entire voltage-clamp ranges examined were diminished (Figure 10). For example, as the hyperpolarizing command voltage from −10 to −90 mV with a duration of 1 s was applied to the tested cell, the peak and sustained components of deactivating I K(erg) were significantly reduced to 283 ± 26 and 16 ± 13 pA (n = 8, p < 0.05) from control values of 336 ± 31 and 28 ± 14 pA (n = 8), respectively. After washout of the compound, peak and sustained I K(erg) was returned to 328 ± 29 and 27 ± 13 pA (n = 7). The mean I-V relationships of peak and sustained I K(erg) acquired in the absence or presence of 10 µM GV-58 were constructed and are hence presented in Figure 10B,C, respectively. However, as cells were exposed to E-4031 (10 µM), an inhibitor of I K(erg) , the I K(erg) evoked from −10 to −100 mV was fully abolished. Therefore, as compared with its effect on I K(A) , the I K(erg) residing in GH 3 cells is mildly suppressed by GV-58 presence. Biomedicines 2022, 10, x FOR PEER REVIEW 15 of 21 Figure 10. Effects of GV−58 on erg−mediated K + current (IK(erg)) in GH3 cells. In these experiments, we kept cells bathed in high-K + , Ca 2+ −free solution containing 1 μM TTX, and the patch pipette was filled up with K + −containing solution. The composition of these solutions is elaborated in Table 1.

Stimulatory Effect of GV-58 on Spontaneous Action Potentials (Aps) Recorded from GH3 Cells
In another set of experiments, we performed current-clamp potential recordings in order to examine any changes in membrane potential of these cells during the absence or presence of GV-58. We kept cells bathed in normal Tyrode's solution containing 1.8 mM CaCl2, and the recording electrode was filled up with K + -containing solution. One minute after cells were exposed to GV-58 at a concentration of 1 or 3 μM, the AP firing recorded under current-clamp configuration was progressively raised (Figure 11). For example, GV-58 (3 μM) resulted in a striking increase in the firing frequency of APs from 0.85 ± 0.05 to 1.52 ± 0.07 Hz (n = 7, p < 0.05). As the compound was removed, the frequency was returned to 0.88 ± 0.06 Hz (n = 6). However, ω-conotoxin MVIID (100 nM) alone had no effect on AP firing. Moreover, the further addition of ranolazine (10 μM), but in continued presence of 3 μM GV-58, was effective in suppressing the frequency of spontaneous APs observed in GH3 cells. It is, thus, possible from the current experiments that GV-58-stimulated increase in AP firing is predominantly linked to its excitatory actions on INa, INa(R), and INa(W) yet not solely by the agonistic effect on CaV channels stated previously [2][3][4][5].

Stimulatory Effect of GV-58 on Spontaneous Action Potentials (Aps) Recorded from GH 3 Cells
In another set of experiments, we performed current-clamp potential recordings in order to examine any changes in membrane potential of these cells during the absence or presence of GV-58. We kept cells bathed in normal Tyrode's solution containing 1.8 mM CaCl 2 , and the recording electrode was filled up with K + -containing solution. One minute after cells were exposed to GV-58 at a concentration of 1 or 3 µM, the AP firing recorded under current-clamp configuration was progressively raised (Figure 11). For example, GV-58 (3 µM) resulted in a striking increase in the firing frequency of APs from 0.85 ± 0.05 to 1.52 ± 0.07 Hz (n = 7, p < 0.05). As the compound was removed, the frequency was returned to 0.88 ± 0.06 Hz (n = 6). However, ω-conotoxin MVIID (100 nM) alone had no effect on AP firing. Moreover, the further addition of ranolazine (10 µM), but in continued presence of 3 µM GV-58, was effective in suppressing the frequency of spontaneous APs observed in GH 3 cells. It is, thus, possible from the current experiments that GV-58-stimulated increase in AP firing is predominantly linked to its excitatory actions on I Na , I Na(R) , and I Na(W) yet not solely by the agonistic effect on Ca V channels stated previously [2][3][4][5]. Biomedicines 2022, 10, x FOR PEER REVIEW 16 of 21

Effect of GV-58 on INa Inherently in NSC-34 Motor Neuron-like Cells
GV-58 was previously reported to enhance spontaneous and evoked activity in the ventral horn of the spinal cord [4]. For these reasons, a final stage of experiments was undertaken to determine whether the INa present in NSC-34 motor neuron-like cells [32] can be vulnerable to any modification by the existence of GV-58. Consistent with the observations made above in GH3 cells, the INa in NSC-34 cells can be enhanced during exposure to GV-58 ( Figure 12A,B). For example, as NSC-34 cells were exposed to 3 μM GV-58, the peak amplitude of INa activated by abrupt depolarizing pulse from −100 to −10 mV was conceivably raised to 587 ± 38 pA (n = 7, p < 0.05) from a control value of 456 ± 27 pA (n = 7). Moreover, as GV-58 was continually present, subsequent addition of riluzole (10 μM) strikingly reduced the magnitude of GV-58-stimulated INa in NSC-34 cells ( Figure 12B). Riluzole has been reportedly demonstrated to suppress INa effectively and to be useful in the management of amyotrophic lateral sclerosis or degenerative cervical myelopathy [33][34][35]. These experimental observations reflect that the ameliorating effect of GV-58 on neuromuscular weakness could partly derive from its stimulation of INa demonstrated herein, although the overarching activation of neuronal CaV channels caused by this compound has been demonstrated [2][3][4][5].

Effect of GV-58 on I Na Inherently in NSC-34 Motor Neuron-like Cells
GV-58 was previously reported to enhance spontaneous and evoked activity in the ventral horn of the spinal cord [4]. For these reasons, a final stage of experiments was undertaken to determine whether the I Na present in NSC-34 motor neuron-like cells [32] can be vulnerable to any modification by the existence of GV-58. Consistent with the observations made above in GH 3 cells, the I Na in NSC-34 cells can be enhanced during exposure to GV-58 ( Figure 12A,B). For example, as NSC-34 cells were exposed to 3 µM GV-58, the peak amplitude of I Na activated by abrupt depolarizing pulse from −100 to −10 mV was conceivably raised to 587 ± 38 pA (n = 7, p < 0.05) from a control value of 456 ± 27 pA (n = 7). Moreover, as GV-58 was continually present, subsequent addition of riluzole (10 µM) strikingly reduced the magnitude of GV-58-stimulated I Na in NSC-34 cells ( Figure 12B). Riluzole has been reportedly demonstrated to suppress I Na effectively and to be useful in the management of amyotrophic lateral sclerosis or degenerative cervical myelopathy [33][34][35]. These experimental observations reflect that the ameliorating effect of GV-58 on neuromuscular weakness could partly derive from its stimulation of I Na demonstrated herein, although the overarching activation of neuronal Ca V channels caused by this compound has been demonstrated [2][3][4][5].

Discussion
The notable findings in this work are that: (a) GH3 cell exposure to GV-58 could differentially increase peak and late components of INa activated by abrupt step depolarization in a concentration-, time-, and state-dependent manner (  Figure 12). The mRNA transcripts for the α-subunit of NaV1.1, NaV1.2, and NaV1.6 were reported to be present in GH3 cells [11]. Together, findings from this study can be

Discussion
The notable findings in this work are that: (a) GH 3 cell exposure to GV-58 could differentially increase peak and late components of I Na activated by abrupt step depolarization in a concentration-, time-, and state-dependent manner ( Figure 1); (b) the I Na activated by brief depolarizing pulse was sensitive to either blockage by tetrodotoxin or stimulation by GV-58, but it failed to be affected by ω-conotoxin MVIID ( Figure 3) (however, no change in the steady-state I-V relation of peak I Na in the presence of GV-58 was noted (Figure 2)); (c) the recovery of I Na inactivation induced with varying interpulse intervals was enhanced in its presence, while I Na decay evoked by a train of depolarizing pulses became slowed ( Figure 4); (d) the decline of peak I Na during rapid repetitive stimuli was slowed in the presence of GV-58 ( Figure 5), while this compound stimulated peak I Na in a tonic and use-dependent manner ( Figure 6); (e) cell exposure to GV-58 was able to enhance I Na(R) and I Na(W) evoked by the descending and ascending ramp pulses, respectively (Figures 7 and 8); (f) the presence of this compound decreased I K(A) together with an increase in inactivation rate of the current, while it mildly reduced I K(erg) measured throughout the entire voltageclamp steps delivered (Figures 9 and 10); (g) GV-58 increased the frequency of spontaneous APs recorded under current-clamp conditions ( Figure 11); and (h) the I Na amplitude in NSC-34 motor neuron-like cells could be enhanced by adding GV-58 ( Figure 12). The mRNA transcripts for the α-subunit of Na V 1.1, Na V 1.2, and Na V 1.6 were reported to be present in GH 3 cells [11]. Together, findings from this study can be interpreted to reflect that the presence of GV-58 can stimulate the amplitude and gating of I Na and that it would engage in the modifications of spontaneous APs in electrically excitable cells (e.g., GH 3 or NSC-34 cells), presuming that similar in vivo results occur.
In the current study, we disclosed that the presence of GV-58 was able to increase recovery of I Na inactivation evoked in response to varying interpulse intervals in a geometricsbased progression; however, it slowed down current inactivation activated during pulsetrain stimulation. Therefore, the post-spike I Na during rapid repetitive stimuli is virtually linked to the prominent slow inactivation in GH 3 cells, suggesting such slow inactivation develops from open channels, and, if recovery from slow inactivation occurs through the open state (conformation), it would be accompanied by a small residual steady current. The presence of GV-58 would be anticipated to enhance the magnitude of post-spike and steady currents, hence raising the occurrence of subthreshold potential [21,24,36,37]. Likewise, the magnitude of ramp-induced I Na(R) and I Na(W) residing in GH 3 cells was expected to become elevated during exposure to GV-58. The simulation by GV-58 of firing frequency recorded under current-clamp experiments is consistent with a previous study made on microelectrode arrays [4]. It is thus reasonable to assume that the GV-58 molecule may have a higher affinity to the open/inactivated state than to the resting (closed) state residing in the Na V channels, although the detailed ionic mechanism of its stimulatory actions on this channel warrants further detailed investigations.
GV-58 has been previously shown to be more potent on N-and P/Q-type Ca 2+ channels with an EC 50 of 6.8 and 9.9 µM, respectively, over L-type Ca 2+ channels (EC 50 > 100 µM) [1]. Additionally, several lines of evidence have been revealed to indicate that GV-58 can stimulate N-and P/Q-type Ca 2+ currents [2][3][4][5]. Pituitary cells have been demonstrated to functionally express two components of Ca V -channel currents found in GH 3 cells [11,38,39]. As such, the question arises as to whether the stimulatory effect of GV-58 on I Na or AP firing observed in GH 3 cells may actually result from the activation of Ca 2+ currents. However, this notion appears to be difficult to reconcile with the current observations revealing that ω-conotoxin MVIID, a blocker of N-and P/Q-type Ca V -channel currents [20], failed to suppress I Na and that further addition of ranolazine, an inhibitor of late I Na [16,19], was able to counteract GV-58-stimulated AP firing. It is also important to notice that GV-58 could not only inhibit the amplitude of I K(A) but also decrease the inactivation time constant of the current, although it mildly suppressed I K(erg) . Therefore, under our experimental conditions, the stimulation of I Na , I Na(R) , and I Na(W) produced by GV-58 tends to occur in a manner largely unlinked to its agonistic actions on Ca V -channel currents. The GV-58 molecule can exert an interaction at the binding site(s) residing preferentially on Na V channels. In this scenario, the accentuation of spontaneous APs caused by the GV-58 existence could result primarily from the accentuation of I Na , including peak or late I Na , I Na(R) , and I Na(W) .
Concentration-dependent stimulation of peak and late I Na with effective EC 50 values of 8.9 and 2.6 µM was, respectively, observed in the presence of GV-58. As cells were exposed to GV-58, the amplitude of peak I Na was increased in combination with a substantial increase in the τ inact(S) value of I Na activation in response to abrupt membrane depolarization. It is also worth noting, from the present observations, that, since EC 50 values detected in this study tend to be lower than EC 50 needed for its activation of Nand P/Q-type Ca V channels [1], the GV-58 actions on I Na and I K(A) reported herein are thus likely to be therapeutically or pharmacologically relevant. Moreover, in our study, in NSC-34 motor neuron-like cells, the exposure to GV-58 not only augmented peak and late I Na but also raised the inactivation time constant of the current. However, whether GV-58-induced amelioration of neuromuscular weakness is pertinent to its stimulatory effect on the amplitude and/or kinetic gating of I Na intrinsically in motor neurons needs to be further delineated. Alternatively, to what extent other structurally related compounds of GV-58 (e.g., ML-50 or GV-05) have any propensity to influence the amplitude and gating of I Na is also worthy of being explored further.
In view of the experimental observations demonstrated herein, the perturbations by GV-58 of transmembrane ionic currents such as I Na and I K(A) tend to be important and, thus, lend credence to the notion that those actions might be in part associated with its actions on the behaviors (e.g., spontaneous APs) in electrically excitable cells [15,36]. The loss of function in I Na (e.g., Na V 1.4-encoded current) has been recently demonstrated to be intimately linked to different neuromuscular disorders (e.g., sodium channel weakness or sodium channel myotonia) [40]. Treatment with 3,4-diaminopyridine base was also revealed to be effective in improving muscle weakness of Lambert-Eaton myasthenia [27]. Awareness thus needs to be stressed in attributing its growing use specifically to the selective agonistic effect on N-and P/Q-type Ca V channels, since previous studies appear to underscore the importance of the increase in I Na and the decrease in I K(A) during the presence of GV-58.

Conflicts of Interest:
The authors declare no competing interests that are directly relevant to this study. A-type K + current I K(erg) erg-mediated K + current I Na voltage-gated Na + current I Na(R) resurgent I Na I Na(W) window I Na Na V channel voltage-gated Na + channel Ran ranolazine Ril riluzole τ inact(S) slow component in the inactivation rate constant TTX tetrodotoxin