The Evidence for Effective Inhibition of I_Na Produced by Mirogabalin ((1R,5S,6S)-6-(aminomethyl)-3-ethyl-bicyclo [3.2.0] hept-3-ene-6-acetic acid), a Known Blocker of Ca_V Channels

Abstract

Mirogabalin (MGB, Tarlige^®), an inhibitor of the α₂δ-1 subunit of voltage-gated Ca²⁺ (Ca_V) channels, is used as a way to alleviate peripheral neuropathic pain and diabetic neuropathy. However, to what extent MGB modifies the magnitude, gating, and/or hysteresis of various types of plasmalemmal ionic currents remains largely unexplored. In pituitary tumor (GH₃) cells, we found that MGB was effective at suppressing the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of the voltage-gated Na⁺ current (I_Na) in a concentration-dependent manner, with an effective IC₅₀ of 19.5 and 7.3 μM, respectively, while the K_D value calculated on the basis of minimum reaction scheme was 8.2 μM. The recovery of I_Na(T) inactivation slowed in the presence of MGB, although the overall current–voltage relation of I_Na(T) was unaltered; however, there was a leftward shift in the inactivation curve of the current. The magnitude of the window (I_Na(W)) or resurgent I_Na (I_Na(R)) evoked by the respective ascending or descending ramp pulse (V_ramp) was reduced during cell exposure to MGB. MGB-induced attenuation in I_Na(W) or I_Na(R) was reversed by the further addition of tefluthrin, a pyrethroid insecticide known to stimulate I_Na. MGB also effectively lessened the strength of voltage-dependent hysteresis of persistent I_Na in response to the isosceles triangular V_ramp. The cumulative inhibition of I_Na(T), evoked by pulse train stimulation, was enhanced in its presence. Taken together, in addition to the inhibition of Ca_V channels, the Na_V channel attenuation produced by MGB might have an impact in its analgesic effects occurring in vivo.

1. Introduction

Mirogabalin (MGB, DS-5565, Tarlige^®, (1R,5S,6S)-6-(aminomethyl)-3-ethyl-bicyclo [3.2.0]hept-3-ene-6-acetic acid), an orally administered gabapentinoid, is a novel, preferentially selective ligand for the α₂δ-1 subunit of voltage-gated Ca²⁺ (Ca_V) channels, and has been used in trials for investigations in the treatment of post-herpetic neuralgia, pain associated with fibromyalgia, and diabetic peripheral neuropathic pain [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. Owing to the considerable research effort, this compound is being investigated for the treatment of peripheral neuropathic pain and fibromyalgia and has demonstrated promising results in patients with diabetic peripheral neuropathy [2,3,4,5,6,9,11,14,15]. Moreover, it has recently been shown to be effective in alleviating anxiety, and in improving cognitive impairments in rats injected repeatedly and intramuscularly with acidic saline [12,13,17]. Based mostly on the potent and long-lasting analgesic effects of MGB, the α₂δ-1 subunit of Ca_V1- and Ca_V2-type voltage-gated Ca²⁺ (Ca_V) channels is considered to play a role in the occurrence of neuropathic pain [3,4,5,7,14,18,19,20]. However, to the best of our knowledge, it is not yet clear whether the presence of MGB has any effects on other types of membrane ionic currents.

The voltage-gated Na⁺ (Na_V) channels are recognized to play a pivotal role in the generation and propagation of action potentials in excitable membranes. The Na_V channel protein contains four homologous domains (D1–D4), each with six transmembrane segments (S1–S6). When rapid depolarization is established, Na_V channels readily go through rapid transitions from the closed (resting) state to the open state and then swiftly to the inactivated state. Genetic defects (i.e., gain-of-function) in Na_V channel inactivation that led to small, sustained Na⁺ currents (I_Na) (i.e., late Na⁺ current [I_Na(L)]) following the occurrence of action potential firing are recognized to have devastating consequences, including neuropathic pain [21,22,23,24].

Nine pore-forming α-subunits (Na_V1.1–1.9) are distributed among excitable mammalian tissues, including central and peripheral nervous systems, and endocrine or heart tissue [25]. The Na_V1.7 and Na_V1.8 subtypes have emerged as key molecules involved in peripheral pain processing and in the development of an increased pain sensitivity associated with inflammation and tissue injury [26,27,28,29,30]. Several activators or inhibitors have been increasingly reported to preferentially modify the late component of the voltage-gated Na⁺ current (i.e., I_Na(L)) [24,31,32,33,34,35,36,37]. However, to date, the issue of whether or how MGB could perturb the magnitude, kinetic gating, or hysteresis of membrane ionic currents (e.g., I_Na) is poorly characterized.

Therefore, in this study, we intended to determine the possible underlying mechanism of MGB actions on the perturbation on different ionic currents (e.g., I_Na) residing in excitable cells (e.g., pituitary GH₃ lactotrophs). The present investigations obtained in this study highlight the evidence showing that MGB can differentially inhibit the transient (I_Na(T)) and late (I_Na(L)) components of I_Na in a concentration-dependent manner in these cells. In addition to the inhibition of Ca_V channels, MGB-mediated interference within the activity of the Na_V channels will converge to act use-dependently on the magnitude, gating, and hysteresis of I_Na in different types of excitable cells.

2. Results

2.1. Inhibitory Effect of MGB on Voltage-Gated Na⁺ Current (I_Na) 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, and the electrode used was filled up with a Cs⁺-containing solution. TEA and CdCl₂ were used to block most of the K⁺ and Ca²⁺ currents, respectively. In this set of whole-cell current recordings, the tested cell was held at −80 mV, a hyperpolarizing pulse of −100 mV was then applied for 30 ms to precede the depolarizing command voltage from −100 to −10 mV, and such a depolarizing step was then imposed to evoke I_Na. Under this experimental protocol, we were able to detect the emergence of an inward current (i.e., inward flux of cations) which displayed the rapidly activating and inactivating time course (Figure 1A). In response to a brief rectangular pulse, this type of transient inward current was sensitive to inhibition or stimulation by tetrodotoxin (TTX, 1 μM) or tefluthrin (Tef, 10 μM), respectively; it has hence been identified as a TTX-sensitive voltage-gated Na⁺ current (I_Na) [24,32,34,35,36,38]. It is of note that one minute after cells were exposed to MGB, the amplitude of peak I_Na (or transient I_Na, [I_Na(T)]) progressively decreased in combination with a concomitant increase in the inactivation time course of the current. For example, the addition of MGB (10 μM) markedly decreased I_Na(T) from 729 ± 33 to 442 ± 23 pA (n = 9, p < 0.05). Additionally, the time constant in the slow component of the current inactivation (τ_inact(S)) was concurrently shortened to 2.2 ± 0.2 ms (n = 9, p < 0.05) from a control of 3.9 ±0.3 ms (n = 9). However, no obvious difference in the fast component of the current inactivation was demonstrated in the presence of MGB. After the washout of the compound, current amplitude returned to 719 ± 29 pA (n = 7, p < 0.05). Moreover, with cell exposure to ranolazine (10 μM), the peak amplitude of I_Na decreased from 732 ± 33 to 321 ± 18 pA (n = 7, p < 0.05).

The relationship between the MGB concentration and the peak (I_Na(T)) or late (I_Na(L)) component of I_Na evoked in response to abrupt membrane depolarizing was further analyzed and tested. In this stage of the experiments, each cell was rapidly stepped from −100 to −10 mV and the I_Na(T) or I_Na(L) measured at different MGB concentrations was collected and then compared. As Figure 1A,B show, the cumulative addition of MGB in the range of 0.3 to 100 μM results in a concentration-dependent reduction in the amplitude of I_Na(T) and I_Na(L). According to a modified Hill equation described in Section 4, the IC₅₀ values for the MGB-mediated inhibition of I_Na(T) and I_Na(L) were computed to be 19.5 and 7.3 μM, respectively. The data, therefore, reflect that MGB exerts a depressant action on the depolarization activated I_Na that is concentration-dependently seen in GH₃ cells, and that the late component of I_Na (I_Na(L)) decreased to a greater extent than the peak component of the current (I_Na(T)) in its presence.

2.2. Kinetic Evaluation of Time-Dependent Block by MGB on I_Na(T) in GH₃ Cells

It was found that increasing the concentration of MGB not only lessened the amplitude of I_Na(T), but also led to an appreciable raise in the magnitude of current inactivation elicited by rapid membrane depolarization (Figure 2A). We quantitatively measured the inactivation time course of I_Na(T) at various MGB concentrations. From the first-order binding scheme described in the Supplementary Information, the relationship of 1/τ_inact(S) versus the MGB concentration became linear (Figure 2B). The forward (on) and backward (off) rate constants were estimated to be 0.124 ms⁻¹μM⁻¹ and 0.102 ms⁻¹, respectively. Consequently, the apparent dissociation constant (i.e., K_D = k₋₁/k₊₁ *) for the binding of MGB to the voltage-gated Na⁺ (Na_V) channel seen in GH₃ cells was estimated to yield 8.2 μM. It is of note that the calculated K_D value has a similarity to the effective IC₅₀ needed for MGB to suppress the amplitude of I_Na(L), although it tends to be measurably smaller than that needed for its reduction in I_Na(T) amplitude.

2.3. Mean Current-Voltage (I-V) Relationship of I_Na(T) Caused by MGB

In the next series of experiments, the I_Na evoked in response to a series of voltage pulses was examined to test whether the presence of MGB exerts any modifications on I_Na(T). In these experiments, when the whole-cell configuration was securely established, the tested cell was held at −80 mV, and voltage pulses ranging between −80 and +10 mV in 10-mV steps were applied for a duration of 30 ms. As depicted in Figure 3A,B, cell exposure to MGB at a concentration of 10 μM led to a progressive reduction in I_Na(T) amplitude, which was concomitantly accompanied by a shortening in the slow component of the inactivation time course of the current (i.e., a decrease in τ_inact(S) value). The mean I-V relationships of I_Na(T) acquired in the control period (i.e., in the absence of MGB) and during cell exposure to 10 μM MGB are illustrated in Figure 3B. For example, when the depolarizing command voltage with a range of −80 to −10 mV was applied to the tested cell, the presence of 10 μM MGB evidently lessened the amplitude of I_Na(T) to 765 ± 88 pA (n = 7, p < 0.05) from a control value of 1749 ± 123 pA (n = 7). After the compound was removed, current amplitude returned to 1728 ± 118 pA (n = 7). Furthermore, the reversal potential of peak I_Na differed between the absence and presence of MGB. The I-V curves obtained in the control period (i.e., when MGB was not present) and during exposure to 10 μM MGB were fitted with a Boltzmann function as described in Section 4. In the control test, G = 39.1 ± 1.2 nS, V_h = −16.5 ± 1.9 mV, and k = 7.1 ± 0.9 (n = 7), while in the presence of 10 μM MGB, G = 22.9 ± 1.1 nS, V_h = −16.7 ± 1.8 mV, and k = 7.2 ± 0.9 (n = 7). These observations indicate that the existence of MGB exerts a depressant action on I_Na(T) intrinsically in GH₃ cells, and that the overall I-V relationship of I_Na(T) is unaltered in its presence, although this drug tends to be selective for sustained over peak I_Na evoked in response to depolarizing command voltages.

In the two-step voltage protocol, the steady-state inactivation curve of I_Na(T), with or without the application of MGB, was constructed and is hence shown in Figure 3C. In control, V_1/2 = −44.8 ± 1.9 mV, and q = 2.9 ± 0.3 e (n = 7), while in the presence of 10 μM MGB, V_1/2 = −65.1 ± 2.1 mV, and q = 3.0 ± 0.3 e (n = 7). Therefore, there was a leftward shift along the voltage axis in the inactivation curve of the current by around 10 mV with no change in the gating charge of the curve.

2.4. Effect of MGB on the Recovery from I_Na(T) Inactivation Evoked during Varying Interpulse Intervals

Next, we examined whether the presence of MGB produces any adjustment on the recovery of I_Na(T) from inactivation, by responding to a two-step voltage protocol in which the interpulse interval increases with a geometric progression (common ratio = 2). In this protocol, a 30 ms step from −80 to −10 mV (prepulse) was firstly applied to the tested cell, and then another 30 ms step to −10 mV (test pulse) was used to inactivate most of the current by varying the duration of the interval between a prepulse and a test pulse. The recovery from current inactivation at the holding potential of −80 mV was then examined at different times with a geometric progression, as presented semi-logarithmically in Figure 4. In the control period (i.e., in the absence of MGB), the peak amplitude of I_Na(T) nearly completely recovered from inactivation when the interpulse duration reached approximately 1 s. The time constants of recovery from current inactivation acquired in the absence and presence of 10 μM MGB were least squares fitted by a single-exponential function with the values of 83.2 ± 2.1 and 156.1 ± 5.9 ms (n = 7, p < 0.05), respectively. The experimental observations indicate that there was a conceivable prolongation in the recovery from inactivation of I_Na(T) as the cells were exposed to MGB.

2.5. Effect of MGB on the Window Component of I_Na (I_Na(W)) Measured from GH₃ Cells

The presence of instantaneous I_Na(W) evoked by the ascending (or upsloping) ramp voltage (V_ramp) was revealed earlier in a variety of excitable cells [37,38,39,40,41]. Next, we explored whether the MGB presence in GH₃ cells could modify the magnitude of I_Na(W) activated in response to the rapid ascending V_ramp. In order to conduct these experiments, the tested cell was voltage-clamped at −80 mV, and we then applied an ascending V_ramp from −110 to +50 mV for a duration of 50 ms to evoke I_Na(W) [37]. As disclosed in Figure 5A,B, within one minute of exposing cells to MGB (10 or 30 μM), the amplitude of I_Na(W) achieved by the 50-ms upsloping V_ramp decreased strikingly. For example, the presence of 10 μM MGB strikingly reduced the area of I_Na(W) measured at the voltage between −40 and +40 mV from 21.2 ± 3.0 to 13.3 ± 2.5 mV·nA (n = 7, p < 0.05). After the drug was removed, current amplitude returned to 20.7 ± 3.1 mV·nA (n = 7). The summary bar graph presented in Figure 5B shows that the addition of MGB is effective in decreasing the I_Na(W) area, and that the subsequent addition of 10 μM tefluthrin (Tef), an insecticide known to be an activator of I_Na [32,42], overcomes the MGB-mediated reduction in the V_ramp-induced I_Na(W) areas. Moreover, the presence of tetrodotoxin (TTX, 1 μM) effectively suppresses the I_Na(W) area, while that of nimodipine, an inhibitor of L-type Ca²⁺ currents, fails to have any effect on it. Tefluthrin was not shown to be an activator of Ca_V channels. The results noted here mean that MGB-mediated suppression of the I_Na(W) area is not mediated through its inhibitory effect on Ca_V channels.

2.6. Suppressive Effect of MGB on Resurgent I_Na (I_Na(R)) Seen in GH₃ Cells

The I_Na(R) was identified earlier in GH₃ cells [32,33], and the magnitude of the current is strongly linked to high-frequency firing observed in Purkinje neurons [43]. In parallel with earlier observations in neurons or endocrine cells [33,44], this type of current is unique in that it is not detectable until the membrane potential is repolarized below 0 mV. In addition to being activated by depolarizing voltage steps rather than by repolarizing voltage steps, I_Na(R) was observed to activate and decay more slowly than I_Na(T). The I_Na(R) is thought to help produce rapid depolarization immediately after an action potential; hence, it is suited either for cells that fire spontaneously at a higher firing rate, or to offer noise modulation in bursting neurons [43,44,45,46]. For these reasons, we additionally investigated whether MGB could exercise any perturbations on such instantaneous current evoked by the descending V_ramp. As the whole-cell configuration was securely established, the 30 ms depolarizing step from −100 to +30 mV followed by a descending (or repolarizing) V_ramp to −80 mV was imposed on the examined cell for a duration of 60 ms. As depicted in Figure 6A,B, the I_Na(R) amplitude activated by such voltage-clamp protocol evidently reduced during GH₃ cell exposure to MGB. For example, the presence of MGB at a concentration of 3 or 10 μM MGB led to a decrease of I_Na(R) at −20 mV from 298 ± 34 pA to 156 ± 28 pA (n = 7, p < 0.05) or 102 ± 15 pA (n = 7, p < 0.05), respectively. The further addition of Tef (10 μM), still in the presence of 10 μM MGB, restored the I_Na(R) amplitude at the same level to 254 ± 32 pA (n = 7, p < 0.05). However, neither further application of nimodipine (1 μM), nor CdCl₂ (0.5 mM), exerted any effects on MGB-inhibited I_Na(R) in the GH₃ cells (155 ± 27 pA [in the presence of 3 μM MGB plus nimodipine], 156 ± 29 pA [in the presence of 3 μM MGB plus CdCl₂], versus 156 ± 28 pA [in the presence 3 μM MGB alone]; n = 7, p < 0.05). It follows, therefore, that the addition of MGB is capable of suppressing I_Na(R) in these cells.

2.7. Effect of MGB on the Hysteretic Behavior of Persistent Na⁺ Current (I_Na(P)) Triggered by Isosceles Triangular Ramp Voltage (V_ramp)

Earlier investigations revealed the capability of the V_hys strength in I_Na(P) to affect electrical behaviors in many types of excitable cells [32,35,36,47,48]. Therefore, we attempted to determine whether and how the presence of MGB could modify the I_Na(P) strength activated in response to a long-lasting upright isosceles triangular V_ramp. In this series of experiments, during the control period or cell exposure to MGB, we voltage-clamped the examined cell at −80 mV, and an upsloping (ascending) limb from −100 to +50 mV, followed by a downsloping (descending) limb back to −100 mV (i.e., upright isosceles triangular V_ramp) was applied to it for a duration of 8 s (Figure 7A). As demonstrated earlier [35], under these experimental conditions, the voltage-dependent hysteresis (V_hys) of I_Na(P) in response to this triangular V_ramp was observed as a striking figure-of-eight (i.e., ∞-shaped) hysteresis in the instantaneous I-V relationship of I_Na(P) (Figure 7A). In other words, there are two distinct loops; that is, the I_Na(P) amplitude at a high- (i.e., in a counterclockwise direction) threshold loop and at a low- (i.e., in a clockwise direction) threshold loop, activated by the upsloping and downsloping limbs of the upright isosceles triangular V_ramp. Of notable interest, as shown in Figure 7B,C, during cell exposure to 3 or 10 μM MGB, the strength of current responding to both rising (i.e., high threshold amplitude) and falling (i.e., low threshold amplitude) limbs of isosceles triangular V_ramp progressively reduced. For example, on the upright isosceles triangular V_ramp, the amplitude activated by the ascending ramp at the level of −10 mV in the presence of 3 or 10 μM MGB decreased, respectively, to 89 ± 10 pA (n = 7, p < 0.05) or 58 ± 8 pA (n = 7, p < 0.05) from a control value of 112 ± 12 pA (n = 7). Likewise, cell exposure to 3 or 10 μM MGB resulted in a measurable decrease in the I_Na(P) amplitude evoked by the descending ramp at −80 mV to 165 ± 27 pA (n = 7, p < 0.05) or 129 ± 19 pA (n = 7, p < 0.05), respectively, from a control value of 232 ± 32 pA (n = 7). As such, the findings from this data enabled us to propose an emergence of V_hys behavior for I_Na(P) activation in response to the upright isosceles triangular V_ramp in GH₃ cells, and that the hysteretic strength of the current was measurably reduced by increasing MGB concentration.

2.8. MGB-Induced Increase in Cumulative Inhibition of I_Na(T) Inactivation

I_Na(T) inactivation was shown to accumulate prior to being activated during repetitive short pulses in previous studies [49,50]. Therefore, additional measurements were taken to study whether the presence of MGB could adjust the inactivation process of the current elicited in a train of depolarizing stimuli. The examined cell was voltage-clamped at −80 mV, and the stimulus protocol, consisting of repetitive depolarization to −10 mV (20 ms in each pulse with a rate of 40 Hz for 1 s), was imposed on it. In keeping with recent observations [50], as depicted in Figure 8A–C, in the control period (i.e., in the absence of MGB), the I_Na(T) inactivation seen in GH₃ cells was evoked by a 1 s repetitive depolarization from −80 to −10 mV with an inactivation time constant of 54.3 ± 4.9 ms (n = 7), i.e., showing a sudden current decay with a single-exponential process. It is of interest that during exposure to MGB, at a concentration of either 3 or 10 μM MGB, the exponential time course of I_Na evoked by the same train of depolarizing pulses shortened to 29.2 ± 3.1 ms (n = 7, p < 0.05) or 12.2 ± 2.5 ms (n = 7, p < 0.05), respectively, in addition to a reduction in I_Na(T) amplitude. As cells were continually exposed to 10 μM MGB, the subsequent addition of Tef (10 μM) reversed the MGB-mediated decrease of current decay with a time constant of 31.2 ± 3.5 ms (n = 7, p < 0.05). Overall, the results indicate that, apart from the decrease in current magnitude, during cell exposure to MGB the decrease in the decaying of I_Na(T) elicited by a 1 s train of depolarizing pulses (i.e., accumulative inactivation of the current) can be enhanced in these cells.

3. Discussion

The promising findings from this study are that: (a) the existence of MGB depresses I_Na in a concentration, time-, state-, use-, and hysteresis-dependent manner as identified in GH₃ cells; (b) this drug resulted in the differential inhibition of I_Na(T) and I_Na(L) activated by short step depolarization with the IC₅₀ value of 19.5 and 7.3 μM, respectively; (c) the K_D value of the MGB-induced increase in current inactivation, estimated according to the first-order binding scheme, was 8.2 μM; (d) MGB did not modify the overall I-V relationship of I_Na(T) but the recovery of I_Na(T) inactivation was prolonged in its presence, while the drug effectively suppressed I_Na(W) and I_Na(R) evoked by ascending or descending V_ramp, respectively; (e) the MGB addition depressed the high- or low-threshold amplitude of I_Na(P) elicited by the isosceles triangular V_ramp at either the upsloping or downsloping limb, respectively; and (f) the cumulative inhibition of I_Na evoked in response to a train of depolarizing pulses was enhanced in the presence of MGB. Collectively, the present observations show that MGB-mediated changes in the magnitude, gating properties, use-dependence, and hysteretic behavior of I_Na would potentially modify the functional activities of excitable cells (e.g., GH₃ cells), presuming that similar in vivo findings are observed.

Perhaps more notable than the issue concerning the magnitude of the MGB-induced reduction in I_Na, is the observation of the non-linear V_hys of I_Na(P) in the control period (i.e., in the absence of MGB) and during cell exposure to MGB, by use of the upright isosceles triangular V_ramp, created through digital-to-analog conversion [47]. During cell exposure to MGB, the peak I_Na(P) activated by the ascending (upsloping) limb of the triangular V_ramp decreased, particularly at the level of −10 mV, while the I_Na(P) amplitude at the descending (downsloping) limb reduced at the level of −80 mV. In this scenario, the instantaneous figure-of-eight (i.e., infinity-shaped: ∞) residing in the V_hys loop that is activated in response to such triangular V_ramp appeared indicating that, as the time goes by during activation, there is a counterclockwise direction in the high-threshold loop (i.e., the relationship of current amplitude as a function of membrane potential), followed by a clockwise direction in the low-threshold loop. In other words, there appears to be two types of V_hys loop, that is, a high-threshold loop with a peak at −10 mV (i.e., activating at a voltage range near the maximal I_Na(T) evoked by brief step depolarization), and a low-threshold loop with a peak at −80 mV (i.e., activating at a voltage near the resting potential). The addition of MGB was able to reduce the V_hys strength of I_Na(P). Therefore, findings from these observations reveal that the triangular V_ramp-induced I_Na(P) undergoes striking V_hys change in the voltage dependence, and that such V_hys loops are subjected to attenuation by adding MGB. On the other hand, it needs to be noted that the V_hys behavior presented here could be strongly linked to the magnitude of sodium background currents as reported previously [21,48]. Further research should be conducted to understand if MGB-mediated changes in V_hys behavior are tightly linked to conformational changes in the voltage sensors of the channel [47].

In this study, the decline of I_Na(T) during a 40 Hz train of depolarizing pulses (i.e., 20 ms pulses applied from −80 to −10 mV at a rate of 40 Hz for a duration of 1 s) becomes pronounced in the presence of MGB, reflecting that there is use-dependence of I_Na(T) during repetitive depolarization as recently demonstrated [50], and that cell exposure to MGB would result in a loss-of-function change caused by the altered, quicker inactivation of the current. Therefore, the MGB-mediated decrease of I_Na(T) is strongly linked to substantial use-dependent facilitation in I_Na(T) during pulse train stimulation.

An earlier report shows that L-type Ca²⁺ channel activation can up-regulate the mRNAs for two different Na_V channels α subunits (Na_V1.2 and Na_V1.3) in GH₃ cells [51]. It is thus postulated that an MGB-induced block of I_Na seen in GH₃ cells could be due, in part, to its inhibitory effect on voltage-gated Ca²⁺ currents that are functionally expressed in excitable cells, including GH₃ cells [52]. However, under our experimental conditions, the voltage-activated inward currents shown herein were either sensitive to stimulation by Tef or subjected to inhibition by TTX and ranolazine. Tef and ranolazine have been reported to be activators or inhibitors of I_Na, respectively [24,32]. In the continued presence of MGB, further addition of Tef could reverse its suppression of I_Na(W) or I_Na(R). It has been previously demonstrated that I_Na(W) and I_Na(R) are responsible for the electrical firing of excitable cells [38,40,43]. Moreover, neither the presence of nimodipine nor CdCl₂ effectively suppressed such inward currents in GH₃ cells. Therefore, it is conceivable that the I_Na (I_Na(L), I_Na(W), I_Na(R) and I_Na(P)) in GH₃ cells is susceptible to being inhibited by MGB, and that its block on I_Na(L) is actually larger than its block on I_Na(T). Moreover, the reduction in I_Na(L) caused by a blocker such as ranolazine can lead to a diminution in Ca²⁺ overload by increasing the driving force for Ca²⁺ extrusion through the Na⁺-Ca²⁺ exchanging process that is functioning in reverse mode (i.e., in a mechanism that operates to extrude Ca²⁺, in exchange for the influx of Na⁺) [53,54]. The mRNA transcripts for the α-subunits of Na_V1.1, Na_V1.2, and Na_V1.6 were demonstrated to be present in GH₃ cells [55]. However, it remains to be determined to what extent MGB can modify I_Na (e.g., Na_V1.7 and Na_V1.8) in dorsal root ganglion neurons, even though such an action could be important for explaining its analgesic potential.

It needs to be emphasized that ranolazine, an inhibitor of I_Na(L), has been demonstrated to be of benefit for peripheral or diabetic neuropathy [56,57,58,59]. This drug has also been revealed to have modifications on changes in peripheral nerve excitability [24,57,58,60,61,62]. As a corollary, it is tempting to anticipate that MGB-mediated alleviation of painful sensation could be partly, if not entirely, attributable to the inhibitory action on different types of I_Na. While the detailed ionic mechanism of its inhibitory action on the Na_V channel is not entirely clear, the MGB molecule may have the propensity to exert a higher effect on the open/inactivated state than on the resting (closed) state residing in the channel, thereby de-stabilizing the open conformation.

Earlier pharmacokinetic studies show that a peak plasma concentration of MGB reached 1000 ng/mL (4.8 μM) one hour after oral administration of 75 mg [63,64]. The effects of MGB on membrane excitability could be likewise dependent on various factors, such as the MGB concentration used, various firing patterns of action potentials [40,43,45], the level of pre-existing resting potential, and in any combinations. It has been noted that some isoforms of the Na_V channel α-subunit were engaged in inflammatory pain states, and they were functionally expressed by somatosensory primary afferent neurons, but not by skeletal or cardiovascular muscle [24,65]. We hitherto demonstrated that the MGB action on excitable membranes is not solely explained by its aberrant use as a blocker on α₂δ subunit of Ca_V channels [1,2]. The activity of Na_V channels in excitable cells may noticeably confer the susceptibility to perturbations by MGB or its structurally similar compounds.

4. Materials and Methods

4.1. Chemicals, Drugs and Solutions Used in This Work

Mirogabalin (MGB, Tarlige^®, DS-5565, (1R,5S,6S)-6-(aminomethyl)-3-ethyl-bicyclo [3.2.0] hept-3-ene-6-acetic acid, C₁₂H₁₉NO₂, CAS No.: 1138245-13-2, purity: ≥98%), was from Cayman Chemical (Genechain Industrial, Kaohsiung, Taiwan), while nimodipine, ranolazine, tefluthrin (Tef), tetraethylammonium chloride (TEA), and tetrodotoxin (TTX) were from Sigma (Merck, Taipei, Taiwan). Unless specified otherwise, cell culture media (e.g., Ham’s F-12 medium), horse serum, fetal calf serum, L-glutamine, and trypsin/EDTA were supplied by HyClone^TM (Merck, Kenilworth, NJ, USA). All other chemicals, such as CdCl₂, CsCl, CsOH, HEPES, and aspartic acid were of the best available quality, mostly at analytical grades.

The ionic composition of extracellular solution (i.e., HEPES-buffered normal Tyrode’s solution) was as follows (in mM): NaCl 136.5, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.53, glucose 5.5, and HEPES 5.5 (pH 7.4 adjusted with NaOH). To record K⁺ currents, the electrode was filled up the internal solution containing (in mM): K-aspartate 130, KCl 20, KH₂PO₄ 1, MgCl₂ 1, EGTA 0.1, Na₂ATP 3, Na₂GTP 0.1, and HEPES 5 (pH 7.2 adjusted with KOH). To measure Na⁺ currents, we replaced K⁺ ions inside the pipette solution with equimolar Cs⁺ ions, and the pH was titrated to 7.2 by adding CsOH. All solutions were prepared using deionized water which was produced by a Milli-Q water purification system (Merck, Kenilworth, NJ, USA).

4.2. Cell Culture

GH₃, a clonal cell line derived from a rat prolactin-secreting pituitary tumor, was acquired from the Bioresources Collection and Research Center (Hsinchu, Taiwan), and the detailed methodology was described earlier [66]. Briefly, cells were maintained in Ham’s F-12 medium (HyClone^TM, Logan, UT, USA) with 15% (v/v) heat-inactivated horse serum, 2.5% (v/v) fetal calf serum, and 2 mM L-glutamine (HyClone^TM, Logan, UT, USA) in a humidified atmosphere of CO₂/air (1:19). The culture medium was changed every 2 to 3 days, and cells were passaged when they reached confluence. Cell viability was evaluated using WST-1 assay (Roche Diagnostics, Taipei, Taiwan). To promote differentiation, cells were transferred to a serum-free, Ca²⁺-free medium. Under these experimental conditions, cells remained 80 to 90% viable for at least 2 weeks. The measurements were conducted 5 or 6 days after cells were grown to 60–80% confluence.

4.3. Electrophysiological Measurements

Before the experiments, we gently dispersed cells with a 1% trypsin/EDTA solution, and an aliquot of cell suspension was directly placed in a recording chamber attached to the fixed-stage of a DM-IL inverted microscope (Leica; Highrise Instrument, Taichung, Taiwan). Cells were immersed at room temperature (20–25 °C) in normal Tyrode’s solution containing 1.8 mM CaCl₂. The electrodes that we used were fabricated from Kimax-51 capillaries (Merck, Taipei, Taiwan) using a PP-83 vertical puller (Narishige; Taiwan Instrument, Tainan, Taiwan), and their tips were thereafter fire-polished with an MF-83 microforge (Narishige; Taiwan Instrument, Tainan, Taiwan). As the electrodes were filled with the different internal solutions described above, their resistance was measured to range between 3 and 5 MΩ, for the purpose of avoiding excessive damage to the cell. Patch-clamp recordings were carried out in whole-cell configuration using either an RK-400 (Bio-Logic, Claix, France) or an Axopatch-200B amplifier (Molecular Devices; Bestgen Biotech, New Taipei City, Taiwan), as described elsewhere [31,37,52,67]. Whole-cell recording was achieved by rupturing the patch of membrane isolated with GΩ sealing by the patch pipet, which brings the cell interior into contact with the pipet interior.

4.4. Data Recordings and Analyses

The signals were simultaneously monitored with a digital oscilloscope (Gould, Chandler, AZ, USA) and a liquid crystal display projector (ViewSonic, Walnut, CA, USA). The data were stored online in a Sony VAIO CS series laptop computer (VGN-CS110E; Tainan, Taiwan), equipped with 1440A digitizer (Molecular Devices). During the measurements with analog-to-digital and digital-to-analog conversion, the latter device was controlled by pCLAMP 10.6 software (Molecular Devices) run on Microsoft Windows 7 (Redmond, WA, USA). The laptop computer used was put on the top of an adjustable Cookskin stand (Ningbo, Zhejiang, China) for convenient manipulation during the experiments. To ensure digitalization, in some sets of measurements, we collected current signals by PowerLab 2/26 acquisition system (ADInstruments; Kuo Yang, Taipei, Taiwan).

To better evaluate the concentration–response curve of MGB-mediated inhibition on the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of I_Na, I_Na were evoked by 30 ms depolarizing pulse to −10 mV from a holding potential of −100 mV, and current amplitudes taken with or without the application of different MGB concentrations (0.3–100 μM) were measured at the start (I_Na(T)) and end pulse (I_Na(L)) of the depolarizing pulse. The concentration required to inhibit 50% of current amplitude was determined according to the three-parameter logistic model (i.e., a modified form of sigmoidal Hill equation) by use of goodness-of-fit assessments:

$$Relative amplitude=\frac{{\left[MGB\right]}^{-n_H}\times \left(1-a\right)}{{\left[MGB\right]}^{-n_H}+I{C}_{50}^{-n_H}}+a$$

where, n_H = the Hill coefficient (i.e., the number bound per side); IC₅₀ = the concentration required for a 50% inhibition); and [MGB] = the MGB concentration. Maximal inhibition (i.e., 1 − a) was approximated in this equation.

The I-V relationship of I_Na(T) with or without addition of MGB was constructed and thereafter 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 membrane potential 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 k and V_h are the gating parameters.

To evaluate the steady-state inactivation of I_Na(T) with or without the existence of MGB, a two-step voltage protocol was created and delivered to the tested cells. A 30 ms conditioning pulse to various membrane potentials preceded the test pulse (30 ms in duration) to −10 mV from a holding potential of −80 mV. The relationship between the normalized amplitude of I_Na(T) and the conditioning potentials was appropriately fitted with another Boltzmann function of the following form:

$$\frac{I}{I_{max}}=\frac{1}{1=exp\left[\left(V-V_{\frac{1}{2}}\right)qF/RT\right]}$$

where I_max is the maximal amplitude of I_Na(T), V_1/2 the voltage at which half-maximal inhibition occurs, q the apparent gating charge of the inactivation curve, F Faraday’s constant, R the universal gas constant, and T the absolute temperature.

The kinetic evaluation of the MGB-mediated effect on I_Na(T) inactivation is provided in the Supplementary Information.

4.5. Curve-Fitting Procedures and Statistical Analyses

Linear or nonlinear curve fitting to experimental data sets in this study was undertaken with the interactive least squares procedure by using different maneuvers, such as Microsoft Excel^®-embedded “Solver” (Microsoft, Redmond, WA, USA) and OriginPro^® 2021 program (OriginLab; Scientific Formosa, Kaohsiung, Taiwan). The averaged results are presented as the mean ± standard error of the mean (SEM) with the sizes of observations (n) indicating the cell numbers from which samples were taken. The paired or unpaired Student’s t-tests between the two groups were applied. When the differences among different groups were encountered, we performed either analysis of variance (ANOVA)-1 or ANOVA-2 with or without repeated measures followed by post-hoc Fisher’s least significant difference test. Statistical analyses were made using the SPSS 20 package (IBM; Tainan, Taiwan). Statistical significance (indicated with * in the figures) was determined at a p value of < 0.05.