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

Abstract

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 [I_Na], A-type K⁺ current [I_K(A)], and erg-mediated K⁺ current [I_K(erg)]) identified from pituitary GH₃ lactotrophs. GH₃ cell exposure to GV-58 enhanced the transient and late components of I_Na with varying potencies; consequently, the EC₅₀ values of GV-58 required for its differential increase in peak and late I_Na in GH₃ cells were estimated to be 8.9 and 2.6 μM, respectively. The I_Na 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 I_Na 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 I_Na. The presence of this compound inhibited I_K(A) magnitude, accompanied by a shortening in inactivation time course of the current; however, it mildly decreased I_K(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 I_Na 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 I_Na in electrically excitable cells, hence modifiying functional activities in these cells.

1. Introduction

GV-58 ((2R)-2-[(6-{[(5-methylthiophen-2-yl)methyl]amino}-9-propyl-9H-purin-2-yl)amino]butan-1-ol), which was developed as a modification of (R)-roscovitine, has been viewed as an opener of N- and P/Q-type Ca²⁺ channels [1,2,3,4,5]. This compound was presumably thought to slow the closing of the voltage-gated Ca²⁺ (Ca_V) channel, resulting in a large increase in total Ca²⁺ entry during motor nerve action potential activity [1]. Its presence was reported to enhance spontaneous and evoked activity from murine ventral horn cultures on microelectrode arrays [4]. Earlier studies have demonstrated that this compound is effective for the management of neuromuscular weakness, such as Lambert–Eaton myasthenic syndrome [3,6,7,8,9]. However, the issue of whether and how other types of ionic currents could be perturbed by this compound remains largely unknown.

It has been established that nine isoforms (i.e., Na_V1.1–1.9 [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 Ca²⁺ 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 GH₃ 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 GH₃ 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 Ca²⁺ (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., GH₃ and NSC-34 cells).

2. Materials and Methods

2.1. Chemicals, Drugs, and Solutions

GV-58 (1402821-41-3, CHEMBL2206242, SCHEMBL17628602, (2R)-2-[(6-{[(5-methylthiophen-2-yl)methyl]amino}-9-propyl-9H-purin-2-yl)amino]butan-1-ol, C₁₈H₂₆N₆OS, https://pubchem.ncbi.nlm.nih.gov/compound/71463101, accessed on 1 December 2022), ω-conotoxin MVIID, and tetrodotoxin (TTX) were acquired from Alomone Labs (Genechain, Kaohsiung, Taiwan), while 4-aminopyridine, E-4031, nimodipine, ranolazine, retinoic acid, riluzole, and tetraethylammonium chloride (TEA) were from Sigma-Aldrich (Merck, Taipei, Taiwan). The stock solution of GV-58 was kept under −20 °C for long-term storage. We obtained culture media (e.g., Ham’s F-12 medium), fetal bovine or calf serum, horse serum, L-glutamine, and trypsin/EDTA from Hyclone^TM (Thermo Fisher Scientific, Tainan, Taiwan), and all other chemicals, including CsCl, CsOH, CdCl₂, and EGTA, were of laboratory grade and supplied from standard sources.

The external solutions and pipette solutions used in this work are illustrated in

Table 1. Composition of normal Tyrode’s solution and the pipette solution used in this study.

Bathing Solution	Purpose or Name	Composition
Bathing solution	Normal Tyrode’s solution	136 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2, 5.5 mM glucose, and 5.5 mM HEPES-NaOH buffer, pH 7.4
Bathing solution	High-K+, Ca2+-free solution	130 mM KCl, 10 mM NaCl, 3 mM MgCl2, 6 mM glucose, 5 mM HEPES-KOH buffer, pH 7.4
Pipette solution	For recordings of K+ current or membrane potential	130 mM K-aspartate, 20 mM KCl, 1 mM KH2PO4, 1 mM MgCl2, 3 mM Na2ATP, 0.1 mM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES-KOH buffer, pH 7.2
Pipette solution	For recordings of Na+ or Ca2+ current	130 mM Cs-aspartate, 20 mM CsCl, 1 mM KH2PO4, 1 mM MgCl2, 3 mM Na2ATP, 0.1 mM Na2GTP, 0.1 mM EGTA, and 5 mM HEPES-CsOH buffer, pH 7.2

.

2.2. Cell Preparations

Pituitary GH₃ somatolactotrophs, supplied by the Bioresource Collection and Research Center ([BCRC-60015, https://catalog.bcrc.firdi.org.tw/BcrcContent?bid=60015, accessed on 1 December 2022], Hsinchu, Taiwan), were maintained in Ham’s F-12 medium, with which 15% heat-inactivated horse serum (v/v), 2.5% fetal calf serum (v/v), and 2 mM L-glutamine were supplemented. NSC-34 neuronal cells were originally produced by fusion of motor neuron-enriched, embryonic mouse spinal cords with mouse neuroblastoma [12,18]. They underwent growth in a 1:1 mixture of DMEM and Ham’s F12 medium with 10% heat-inactivated fetal bovine serum (v/v) and 1% penicillin-streptomycin. To slow cell proliferation and enhance their maturation towards a differentiated state, before confluence, we maintained NSC-34 cells in 1:1 DMEM plus Ham’s F-12 medium supplemented with 1% fetal bovine serum and 1 μM retinoic acid. GH₃ and NSC-34 cells were grown in monolayer culture in 50 mL plastic culture flasks in a humidified environment of CO₂/air (1:19). Subcultures were made by trypsinization (0.025% trypsin solution [HyClone^TM] containing 0.01% sodium N,N,-diethyldithiocarbamate and EDTA). We conducted the electrophysiological measurements when cells reached 60–79% confluence (usually 5–7 days).

2.3. Electrophysiological Measurements

During the few hours before the experiments, we gently dissociated GH₃ 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 (Section 3.3 and Section 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).

2.4. 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 Ca²⁺-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 GH₃ cells were determined by fitting the modified Hill equation. That is,

$$Percentage increase (\%)=\left(E_{max}\times {\left[GV-58\right]}^{n_H}\right)/\left(E{C}_{50}^{n_H}+{\left[GV-58\right]}^{n_H}\right)$$

where [GV-58] is the GV-58 concentration applied, n_H the Hill coefficient, EC₅₀ 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:

$$\frac{I}{I_{max}}=\frac{G}{1+exp\left[-\left(V-V_h\right)/k\right]}\times \left(V-E_{rev}\right)$$

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.

2.5. 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.

3. Results

3.1. Stimulatory Effect of GV-58 on Voltage-Gated Na⁺ Current Measured from Pituitary GH₃ Cells

For the first stage of measurements, we kept cells bathed in Ca²⁺-free Tyrode’s solution which contained 10 mM tetraethylammonium chloride (TEA) and 0.5 mM CdCl₂. TEA and CdCl₂ were used to block most of the K⁺ and Ca²⁺ 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).

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, GH₃ cell exposure to GV-58 resulted in a concentration-dependent rise in the amplitude of peak and late I_Na. The EC₅₀ 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 GH₃ cells and that the late component of I_Na increases to a greater extent than the peak component of the current in its presence.

3.2. 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 Ca²⁺ currents (e.g., N- and P/Q-type Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ currents is unlikely to be linked to GV-58-stimulated I_Na found in GH₃ cells.

3.3. 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.

3.4. 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.

3.5. GV-58 Effect on I_Na 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 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 pulse-train 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.

3.6. 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 Ca²⁺-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-58-stimulated I_Na tends to be composed of two components, namely, tonic and use-dependent components.

3.7. Stimulatory Effect of GV-58 on Resurgent I_Na (I_Na(R)) Seen in GH₃ Cells

The I_Na(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, 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 CdCl₂ (0.5 mM) failed to exert any effects on GV-58-stimulated I_Na(R) in GH₃ cells. Ranolazine was previously demonstrated to be an inhibitor of late I_Na [16,19].

3.8. Effect of GV-58 on the Window Component of I_Na (I_Na(W)) Recorded from GH₃ 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 GH₃ 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 voltage-clamped 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.

3.9. Inhibitory Effect of GV-58 on A-Type K⁺ Current (I_K(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., I_K(A)) were also further examined with respect to possible modifications of GV-58 on them. In these experiments, we bathed cells in Ca²⁺-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 (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 GH₃ 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 GH₃ cells.

3.10. 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 GH₃ cells. This set of measurements was conducted in cells bathed in high-K⁺, Ca²⁺-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 GH₃ cells is mildly suppressed by GV-58 presence.

3.11. 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 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].

3.12. 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 GH₃ 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].

4. Discussion

The notable findings in this work are that: (a) GH₃ 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 (Figure 7 and Figure 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 voltage-clamp steps delivered (Figure 9 and Figure 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_V1.1, Na_V1.2, and Na_V1.6 were reported to be present in GH₃ 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., GH₃ 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 geometrics-based progression; however, it slowed down current inactivation activated during pulse-train stimulation. Therefore, the post-spike I_Na during rapid repetitive stimuli is virtually linked to the prominent slow inactivation in GH₃ 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 GH₃ 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 Ca²⁺ channels with an EC₅₀ of 6.8 and 9.9 μM, respectively, over L-type Ca²⁺ channels (EC₅₀ > 100 μM) [1]. Additionally, several lines of evidence have been revealed to indicate that GV-58 can stimulate N- and P/Q-type Ca²⁺ currents [2,3,4,5]. Pituitary cells have been demonstrated to functionally express two components of Ca_V-channel currents found in GH₃ 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 GH₃ cells may actually result from the activation of Ca²⁺ 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 EC₅₀ 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 EC₅₀ values detected in this study tend to be lower than EC₅₀ needed for its activation of N- and 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_V1.4-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.