Effective Perturbations by Phenobarbital on I_Na, I_K(erg), I_K(M) and I_K(DR) during Pulse Train Stimulation in Neuroblastoma Neuro-2a Cells

Abstract

Phenobarbital (PHB, Luminal Sodium^®) is a medication of the barbiturate and has long been recognized to be an anticonvulsant and a hypnotic because it can facilitate synaptic inhibition in the central nervous system through acting on the γ-aminobutyric acid (GABA) type A (GABA_A) receptors. However, to what extent PHB could directly perturb the magnitude and gating of different plasmalemmal ionic currents is not thoroughly explored. In neuroblastoma Neuro-2a cells, we found that PHB effectively suppressed the magnitude of voltage-gated Na⁺ current (I_Na) in a concentration-dependent fashion, with an effective IC₅₀ value of 83 µM. The cumulative inhibition of I_Na, evoked by pulse train stimulation, was enhanced by PHB. However, tefluthrin, an activator of I_Na, could attenuate PHB-induced reduction in the decaying time constant of I_Na inhibition evoked by pulse train stimuli. In addition, the erg (ether-à-go-go-related gene)-mediated K⁺ current (I_K(erg)) was also blocked by PHB. The PHB-mediated inhibition on I_K(erg) could not be overcome by flumazenil (GABA antagonist) or chlorotoxin (chloride channel blocker). The PHB reduced the recovery of I_K(erg) by a two-step voltage protocol with a geometrics-based progression, but it increased the decaying rate of I_K(erg), evoked by the envelope-of-tail method. About the M-type K⁺ currents (I_K(M)), PHB caused a reduction of its amplitude, which could not be counteracted by flumazenil or chlorotoxin, and PHB could enhance its cumulative inhibition during pulse train stimulation. Moreover, the magnitude of delayed-rectifier K⁺ current (I_K(DR)) was inhibited by PHB, while the cumulative inhibition of I_K(DR) during 10 s of repetitive stimulation was enhanced. Multiple ionic currents during pulse train stimulation were subject to PHB, and neither GABA antagonist nor chloride channel blocker could counteract these PHB-induced reductions. It suggests that these actions might conceivably participate in different functional activities of excitable cells and be independent of GABA_A receptors.

1. Introduction

Phenobarbital (PHB, phenobarbitone, phenobarb), also known by the trade name Luminal Sodium^®, is a medication belonging to a group known as barbiturates. PHB has long been recognized to be an anticonvulsant and a hypnotic because it can facilitate synaptic inhibition in the central nervous system by acting on the γ-aminobutyric acid (GABA) type A (GABA_A) receptors [1,2,3,4,5,6,7]. GABA_A receptor is a ligand-gated chloride ion channel, which is the most common inhibitory channel in the brain. Pentobarbital also belongs to barbiturates and is similar to phenobarbital. It is recently disclosed to suppress neurogenic inflammation and exert neuroprotective and even anti-neoplastic activities by modifying membrane ion channels other than chloride channels [8,9,10]. Membrane ion channels mediate the excitability of membrane potentials, which is essential to the excitable cells, ex, the neurons. Several neurological diseases, including epilepsy, migraine, movement disorders, or muscular disorders, are caused by the dysfunctions of membrane ion channels [1,11]. Thus, we are curious whether PHB might also exert additional actions on other membrane ionic currents in addition to GABA_A receptor-mediated chloride currents.

In this study, membrane Na⁺ and K⁺ currents are of interest. As we know, the voltage-gated Na+ (NaV) channels are important for generating and propagating action potentials (APs) in excitable cells [12]. Upon rapid depolarization, Na_V channels can go through rapid transitions from the closed (resting) state to the open state and then swiftly change to the inactivated state [12,13]. The inactivation of voltage-gated Na⁺ current (I_Na) has been demonstrated to accumulate before being stimulated during repetitive short depolarizing pulses [14,15]. Several kinds of K⁺ currents were included. First is the erg (ether-à-go-go-related gene) -mediated K⁺ current (I_K(erg)) gated by voltage-dependent K⁺ (K_V) channels of EAG (ether-à-go-go) family. I_K(erg) has been known to be intrinsic in cardiac cells or variable types of excitable cells, such as neuroblastoma or neuroendocrine cells. The erg K_V channels have peculiar gating kinetics, i.e., rapid inactivation and slow deactivation kinetics. These characteristics are essential in maintaining resting potential and modifying the subthreshold excitability or rhythmic oscillations [16,17,18,19,20,21]. The time-dependent decay of I_K(erg) evoked by the envelop-of-tail test has also been modified by different small molecules (e.g., azimilide, opioid agonists, and isoplumbagin) [21,22,23,24]. Therefore, it is worthy of investigating to what extent PHB itself can modify the magnitude and gating of I_K(erg).

The KCNQ2, KCNQ3, or KCNQ5 gene is viewed to encode the core subunit of the K_V7.2, K_V7.3, or K_V7.5 channel, respectively. The activity of these K_V channels can generate the macroscopic M-type K⁺ current (I_K(M)). The biophysical properties of I_K(M) are known to exhibit current activation in response to low-threshold voltage and to display a slowly activating and deactivating time course of the current [25,26]. Furthermore, the magnitude of I_K(M) can potentially regulate the availability of Na_V channels during prolonged high-frequency firing [27]. Notable studies have also shown that the presence of either the benzodiazepine activator or cannabidiol could activate K_V7-encoded currents [28,29]. Therefore, further investigations are warranted to delineate whether PHB has any perturbations on this type of K⁺ currents.

The K_V channels play a role in determining the membrane excitability associated with delayed-rectifier K_V channels. The activity of K_V3 (KCNC) or K_V2 (KCNB) channels and the magnitude of delayed-rectifier K⁺ current (I_K(DR)) are correlated with AP firing in many cell types [18,26,30]. This type of I_K(DR) activated during pulse train (PT) stimulations have the propensity to induce the resurgent K⁺ tail current, which is proposed to serve as a negative-feedback mechanism for the closure of K_V channels during high-frequency firing [31]. However, the extent to which PHB can modify the magnitude of I_K(DR), especially during pulse train (PT) stimuli, still remains unclear.

In the current study, the electrophysiological effects of PHB on these membrane ion currents were extensively investigated in Neuro-2a cells. Neuro-2a cells have been previously reported to express functional GABA_A receptors with barbiturate binding sites [32]. These membrane ion currents, including I_Na, I_K(erg), I_K(M), and I_K(DR), were explored with a single voltage-clamp pulse or pulse train stimulation and were synergistically inhibited by PHB. This PHB-induced synergistic inhibition of ionic currents may partially contribute to the underlying mechanisms through which PHB or other barbiturates affect the electrical behaviors of excitable cells in cell culture and in vivo.

2. Materials and Methods

2.1. Chemicals, Drugs, and Solutions

Phenobarbital (PHB, Luminal Sodium^®, Solfoton^®, Tedral^®, phenobarbitone, phenobarbitol, 5-ethy-5-pheny-1,3-diazinane-2,4,6-trione [IUPAC name], 5-ethyl-5-phenylbarbituric acid, C₁₂H₁₂N₂O₃), flumazenil (FLM), tetraethylammonium chloride (TEA) and tetrodotoxin (TTX) were purchased from Sigma-Aldrich (Genechain, Kaohsiung, Taiwan). Thiopental sodium (Pentothal^®) was acquired from SCI Pharmatech (Taoyuan, Taiwan), while midazolam was from Nan Kuang Pharmaceutical (Tainan, Taiwan). Chlorotoxin was kindly provided by Professor Dr. Woei-Jer Chuang (Department of Biochemistry, National Cheng Kung University Medical College, Tainan, Taiwan) [21].

For cell preparations, culture media, fetal bovine serum, L-glutamine, and trypsin/EDTA were supplied by HyClone^TM (Thermo Fisher, Tainan, Taiwan). All other chemicals were acquired from regular commercial chemicals and of reagent grade. In the current study, reagent water was obtained from a Milli-Q ultrapure water purification system (Millipore; Merck, Tainan, Taiwan).

2.2. Cell Preparation

Neuro-2a (N2a), a clonal cell line originally derived from mouse neuroblastoma, was purchased from the Bioresources Collection and Research Center ([BCRC-60026, https://catalog.bcrc.firdi.org.tw/BcrcContent?bid=60026 (accessed on 10 Jan 2022)], Hsinchu, Taiwan). Neuro-2a cells, originally derived from the American Type Culture Collection (ATCC^® [CCL-131^TM]; Manassas, VA, USA), has been used as electrically excitable cells in many studies of electrophysiology and pharmacology [11,33,34,35,36]. Cells were cultured in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1.5 g/liter sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate in a humidified atmosphere of CO₂/air (1:19) at 37 °C [21,37,38]. Subcultures were made by trypsinization (0.025% trypsin solution [HyClone^TM] containing 0.01% sodium, N,N,-diethyldithiocarbamate and EDTA). Electrophysiological measurements were commonly conducted when cells reached 50–70% confluence (usually 5–7 days) [21].

2.3. Electrophysiological Measurements

In the few hours before the experiments, we dispersed Neuro-2a cells with a 1% trypsin/EDTA solution, and a few drops of cell suspension (10⁶/mL) was quickly transferred to a custom-made chamber firmly mounted on the working stage of a DM-IL inverted microscope (Leica; Major Instruments, Kaohsiung, Taiwan). Cells were then bathed at room temperature (20–25 °C) in normal Tyrode’s solution, whose composition is described above. Before each experiment, cells were allowed to settle on the chamber’s bottom. The patch pipettes were pulled from Kimax^®-51 borosilicate glass tube (#DWK34500–99; Kimble^®, Merck, Tainan, Taiwan) and were further polished to reach their resistances ranging between 3 and 5 MΩ. During the recordings, the electrodes were mounted in an air-tight holder, which had a suction port on the side, and a silver-chloride wire was used to make contact with the internal electrode solution [39]. We recorded varying types of ionic currents (i.e., I_Na, I_K(erg), I_K(M), and I_K(DR)) with the whole-cell mode of a modified patch-clamp technique by using either an Axoclamp-2B (Molecular Devices, Sunnyvale, CA) or an RK-400 amplifier (Bio-Logic, Claix, France), as described elsewhere [19,21,26,40]. The liquid junction potentials that occur when the composition of the pipette internal solution differed from that in the bath were zeroed shortly before giga-Ω formation was made, and the whole-cell data were corrected [21]. As pulse train (PT) stimulation was applied to the tested cell, we used an Astro-Med Grass S88X dual output pulse stimulator (Grass; KYS Technology, Tainan, Taiwan).

2.4. Data Recordings

The data were stored online in an ASUSPRO-BU4011LG laptop computer (ASUS, Tainan, Taiwan) at the sampling rate of 10 kHz. The computer was equipped by a Digidata^® 1440A acquisition device (Molecular Device; Bestgen Biotech, New Taipei City, Taiwan), through which analog-to-digital and digital-to-analog conversions were controlled by pCLAMP^® 10.6 software (Molecular Devices). Current signals were low pass filtered at 3 kHz. We analyzed off-line the signals acquired during the experiments by use of different analytical tools, including LabChart^TM 7.0 program (AD Instruments; Gerin, Tainan, Taiwan), OriginPro^® 2021 (OriginLab Corp.; Scientific Formosa, Kaohsiung, Taiwan), and custom-made macros built in Excel^® 2022 (Redmond, DC, USA). We created various voltage-clamp protocols used in this work from pCLAMP^® 10.6 and, through digital-to-analog conversion, different waveforms were used to investigate the current versus voltage (I-V) relationship, the steady-state inactivation curve, or the recovery of current inactivation for specific ionic currents (i.e., I_Na or I_K(erg)) [12,21].

2.5. Whole-Cell Data Analyses

In order to evaluate the percentage inhibition of PHB on the I_Na amplitude, each tested cell was 30 ms depolarized from −100 to −10 mV, and current magnitudes during cell exposure to different PHB concentrations were measured and compared at the start of the voltage pulse. The concentration of PHB required to suppress 50% of the peak component of I_Na activated in response to short depolarizing pulse was thereafter determined using a Hill function:

$$y=\raisebox{1ex}{$E_{max}$}\!\left/ \!\raisebox{-1ex}{$\left\{1+\left(I{C}_{50}^{n_H}/{\left[PHB\right]}^{n_H}\right)\right\}$}\right.$$

where y = percentage inhibition (%); [PHB] = the PHB concentration applied; n_H = the Hill coefficient; IC₅₀ = the concentration required for a 50% inhibition of I_Na; E_max = PHB-induced maximal inhibition of peak I_Na.

2.6. Curve-Fitting Approximations and Statistical Analyses

Curve parameter estimation was made by a non-linear or linear fitting routine with a least-squares minimization procedure, in which the “Solver” add-in bundled with Excel^® 2021 (Microsoft, Redmond, WA, USA) was employed [41]. The results are presented as the mean ± standard error of the mean (SEM). The sizes of independent samples (n) indicated the cell number from which the investigation was performed. The statistical significance between the two groups was analyzed by using paired or unpaired Student’s t-test, while as the difference among more than two groups was needed, post hoc least-significance tests were further performed. The statistical analyses were performed by using SPSS 20.0 software (IBM Corp., Armonk, NY, USA). A statistical significance was considered when p < 0.05.

3. Results

3.1. Effects of PHB on the Amplitude of Voltage-Gated Na⁺ Current (I_Na) in Neuro-2a Cells

In the beginning, the I_Na amplitude activated in response to rapid membrane depolarization was tested in different concentrations of PHB. Cells were placed in Ca²⁺-free, Tyrode’s solution, which contained 10 mM tetraethylammonium chloride (TEA) and 0.5 mM CdCl₂. TEA and CdCl₂ are recognized to block voltage-gated K⁺ and Ca²⁺ channels, respectively. Upon the short depolarizing pulse from −100 to −10 mV, the peak amplitude of I_Na with a rapidly activating and inactivating time course was decreased at the exposure to 30 and 100 µM PHB (Figure 1A). For example, in the presence of 100 µM PHB, I_Na amplitude was profoundly reduced to 812 ± 25 pA (n = 8, p < 0.05) from a control value of 1867 ± 78 pA (n = 8, Figure 1A). The current amplitude was returned to 833 ± 29 pA (n = 8) after the removal of PHB. The time constants of I_Na activation, and fast and slow components of current inactivation in the control period (i.e., no PHB) were respectively 0.61 ± 0.01, 0.91 ± 0.01, and 1.93 ± 0.03 ms (n = 8), while those of activation, and fast and slow components of inactivation in the presence of PHB were respectively 0.61 ± 0.01, 0.92 ± 0.01, and 1.94 ± 0.03 ms (n = 8, p > 0.05). It indicates no obvious change in activation or inactivation time course of I_Na activated by abrupt membrane depolarization was demonstrated as cells were exposed to PHB.

The relationship between the PHB concentrations and the peak I_Na was further constructed. As demonstrated in Figure 1B, the I_Na amplitudes obtained at different PHB concentrations were collated and then estimated. The cumulative addition of PHB in the range between 3 µM and 1 mM resulted in a concentration-dependent reduction in the peak amplitude of I_Na. According to a modified Hill equation stated in Materials and Methods, the IC₅₀ value for the PHB-mediated inhibition of peak I_Na was optimally yielded to be 83 µM. The data, therefore, reflect that the PHB can exert a depressant action on the depolarization-activated I_Na in a concentration-dependent fashion in Neuro-2a cells.

3.2. PHB-Induced Increase in Cumulative Inhibition of I_Na Inactivation

High-frequency action potential transmission is important for rapid information processing in the central nervous system. I_Na inactivation has been previously shown to accumulate prior to being activated during repetitive pulse train (PT) stimulation [14,15,40,42]. Therefore, further experiments were performed to explore whether PHB could modify the inactivation process of I_Na elicited by the PT stimuli. The PT stimuli were induced by repetitive PT depolarizations to −10 mV (20 ms in each pulse with a rate of 40 Hz for 1 s) when the cell was held at −80 mV. In Figure 2(Aa),B, the I_Na inactivation was evoked in Neuro-2a cells by a 1 s PT stimulus from −80 to −10 mV with the single-exponential time constant (τ) of 104 ± 28 ms (n = 7) in the control period (i.e., no PHB). It indicates a considerable slowing in current inactivation during PT stimulation. When cells exposure to PHB (30 or 100 µM), the exponential time course of I_Na evoked by the same PT stimulations was strikingly shortened to 81 ± 22 ms (n = 8, p < 0.05) or 45 ± 14 ms (n = 8, p < 0.05), respectively (Figure 2(Ab),B), in addition to a profound decrease in I_Na amplitude in response to rapid membrane depolarization. Furthermore, tefluthrin (Tef, 10 µM) could partially reverse the PHB-mediated reduction of current decay with a τ value of 87 ± 23 ms (n = 8, p < 0.05, Figure 2B). Tef is a synthetic insecticide known to activate I_Na [43,44,45].

3.3. Effects of PHB on erg-Mediated K⁺ Current (I_K(erg)) Residing in Neuro-2a Cells

We continued to explore the possible perturbations of PHB on the magnitude of I_K(erg). In order to amplify I_K(erg), cells were placed in a high-K⁺, Ca²⁺-free solution containing 1 µM TTX. The recording electrode was filled up with a K⁺-enriched solution [18,19,46]. The different command voltages ranging between −120 and −10 mV for 1 s were imposed to induce hyperpolarization-activated I_K(erg) when the tested cell was held at −10 mV (Figure 3A) [18,19,21,26]. The average current-versus-voltage (I-V) relationship of the peak (filled symbols) or sustained (open symbols) component of I_K(erg) in the absence (control) or presence of 300 µM PHB was constructed, respectively (Figure 3B,C). For example, upon membrane hyperpolarization from −10 mV, PHB decreased the peak I_K(erg) from a control value (i.e., no PHB) of 686 ± 156 pA (n = 8) to 343 ± 112 pA (n = 8, p < 0.05) at the level of −60 mV and decreased the sustained I_K(erg) from 526 ± 82 pA (n = 8) to 222 ± 88 pA (n = 8, p < 0.05). The presence of PHB (300 µM) could suppress both the peak and sustained component of deactivating I_K(erg) throughout the entire measurement (Figure 3C).

We further investigated whether PHB-induced inhibition of I_K(erg) was associated with GABA-mediated inhibition or not. FLM is an antagonist of GABA_A receptors, while chlorotoxin is a blocker of Cl- channels. In the presence of PHB (300 µM), neither FLM nor chlorotoxin could overcome the PHB-mediated inhibition of I_K(erg) activated by long-lasting membrane hyperpolarization (Figure 3D). The results suggested that PHB-induced inhibition of I_K(erg) in these cells is unlinked to its propensity to interact with GABA_A receptors, as reported previously in other cell types [7,9,33,47,48,49,50].

3.4. Modification by PHB on the Magnitude of I_K(erg) Activated by PT Stimulation

We further explored if PHB could modify the extent of I_K(erg) activated in the PT hyperpolarizing stimuli. The stimulus protocol, consisting of repetitive hyperpolarization to −110 mV (40 ms in each pulse with a rate of 20 Hz for 1 s), was imposed over the tested cells held at −10 mV. In the absence of PHB (control), the 1 s repetitive hyperpolarization from −10 to −110 mV evoked the I_K(erg) decay with a decaying τ of 147 ± 22 ms (n = 7) (Figure 4(Aa)). Upon exposure to 300 µM PHB, the τ value in the exponential time course of decaying I_K(erg) evoked by the same train of hyperpolarizing pulses was shorted to 87 ± 11 ms (n = 7, p < 0.05) (Figure 4(Ab)), and the I_K(erg) amplitude was reduced, too (Figure 4B). E-4031 is an inhibitor of I_K(erg) [18,51], and E-4031 could further diminish the I_K(erg) amplitude in the presence of 300 µM PHB (Figure 4B). These results indicate that, apart from the reduction in I_K(erg) magnitude, the decrease in the decaying of I_K(erg) elicited by a 1 s PT hyperpolarizing pulse (i.e., accumulative inactivation of the current) can be enhanced by PHB in these cells.

3.5. Slowing in Recovery from I_K(erg) Block Caused by PHB in Neuro-2a Cells

We continued to explore if PHB could lead to any modifications on recovery from the block of I_K(erg). The recovery from the current block was conducted by using a two-step voltage-clamp protocol in which the interval of depolarizing command pulses (i.e., conditioning pulse) varies with a geometrics-based progression. After each conditioning pulse, a hyperpolarizing pulse stepped back to −110 mV for 500 ms was applied to evoke deactivating I_K(erg). As the duration of the conditioning pulse was set at 2048 ms, the amplitude of the tail current (i.e., deactivating I_K(erg)) activated by hyperpolarizing step from −10 to −110 mV with a duration of 500 ms was taken as 1.0. The relative amplitude of I_K(erg) with different duration of conditioning pulse was measured and then compared. In the absence of PHB (control), the peak amplitude of deactivating I_K(erg) was fully restored from the block when the pulse duration was set at 2048 ms or above (Figure 5(Aa),B). Interestingly, the time course of recovery from the current block was appropriately fitted by a single exponential, and the τ values could be derived. The τ values were 387 ± 12 ms (n = 7) in the absence of PHB and 823 ± 23 ms (n = 7) in the presence of PHB. As a result, PHB produces a considerable lengthening in the recovery from the block of deactivating I_K(erg) in Neuro-2a cells.

Modification by PHB of the Time Course of I_K(erg) Evoked by the Envelope-of-Tail Test

Earlier investigations have demonstrated a time-dependent change in I_K(erg) activation during the envelope-of-tail test [21,22,23,24,52]. Here, we then analyzed an inward activating and deactivating I_K(erg) evoked by varying durations of conditioning pulse (from 4 to 2048 ms with a geometrics-based progression) to −10 mV from −110 mV, and the relationship of the relative amplitude versus the pulse duration was established in Figure 5C. Like earlier investigations [21,24], the envelope-of-tail test tailored for the activation of I_K(erg) seen in Neuro-2a cells exhibits a time-dependent exponential decay in the ratio of the relative amplitude (i.e., I_act/I_deact) evoked during the pulse durations between 4 and 2048 ms in a geometrics-based progression. When Neuro-2a-cell exposure to 300 µM PHB, the τ value of I_K(erg) activated by the envelope-of-tail method was significantly reduced to 64 ± 3 ms (n = 7, p < 0.05) from a control value of 112 ± 11 ms (n = 7). It is conceivable that PHB has the propensity to reduce the time course of I_K(erg) activation evoked during the envelope-of-tail voltage protocol residing in Neuro-2a cells.

3.6. Modification by PHB of M-Type K⁺ Current (I_K(M)) in Neuro-2a Cells

Next, we continued examining if PHB could modify the magnitude of I_K(M) in Neuro-2a cells. Cells were placed in a high-K⁺, Ca²⁺-free solution, and the recording electrode was filled up with K⁺-enriched solution. Upon membrane depolarization from −50 to −10 mV, PHB-treated cells exerted an inhibitory effect on I_K(M) amplitude (Figure 6A,B). This PHB-mediated inhibition of I_K(M) amplitude could not be reversed by adding FLM (10 µM) or chlorotoxin (1 µM) (Figure 6B). Thus, unlike the stimulatory action of benzodiazepine derivative or cannabidiol on K_V7-encoded currents, PHB exerts a depressant action of I_K(M) in Neuro-2a cells. However, the inactivating properties of I_K(M) shown in Figure 6A could be due to the interference by other types of delayed-rectifier K+ currents. The averaged I-V relationships of I_K(M) with or without PHB (300 μM) were made, and the results are shown in Figure 6C.

3.7. Modification by PHB on I_K(M) Inactivation during PT Stimulation

A recent study has demonstrated the capability of I_K(M) to maintain the availability of Na_V channels during prolonged high-frequency firing [27]. Therefore, we further examined if the PHB could modify the extent of I_K(M) during PT stimulation in Neuro-2a cells. The results showed that PHB (100 µM or 300 µM) obviously decreased the amplitudes of activating and deactivating I_K(M) during 1 s PT stimulation (Figure 7A–C). For example, PHB (300 µM) decreased the amplitudes of activating I_K(M) to 165 ± 21 pA (n = 7, p < 0.05, Figure 7B) and the amplitudes of activating I_K(M) to 459 ± 195 pA (n = 7, p < 0.05, Figure 7C). These disclosed that PHB-mediated inhibition of I_K(M) remained efficacious upon high PT stimulation. It is conceivable that the availability of Na_V channels during high-frequency firing in unclamped cells became further retarded during the exposure to PHB, despite its ability to suppress the I_Na amplitude directly, as described above.

3.8. Inhibitory Effect of PHB on Delayed-Rectifier K⁺ Current (I_K(DR)) Residing in Neuro-2a Cells

The I_K(DR) has been previously revealed to be suppressed by pentobarbital, another barbiturate, as well as to display the resurgent K⁺ tail currents during high PT stimulations [50,53] We additionally explored if the PHB could modify the magnitude of I_K(DR) in these cells. In this series of experiments, cells were placed in Ca²⁺-free, Tyrode’s solution, which contained 1 µM TTX and 0.5 mM CdCl₂, and the recording electrode used was filled up with K⁺-enriched solution. Under the voltage-clamp current recordings, the tested cell was held at −50 mV, and a series of voltage pulses ranging between −60 and +50 mV was imposed over it. In Figure 8A, upon the presence of 300 µM PHB, the I_K(DR) amplitude was clearly suppressed, especially at the voltages above −10 mV. The average I-V relationship of I_K(DR) is illustrated in Figure 8B.

3.9. PHB-Induced Increase in Cumulative Inhibition of I_K(DR) Inactivation in Neuro-2a Cells

In the last series of measurements, we wanted to investigate if PHB can modify the time course of I_K(DR) in response to long-lasting PT stimulation intrinsically in Neuro-2a cells. Under the control condition (i.e., no PHB), a single 10 s depolarizing step from −50 to +50 mV resulted in an exponential decline with a time constant of 2.12 ± 0.08 s (n = 8) (Figure 9A). In contrast, the time constant for 10 s PT stimulation to +50 mV, each of which lasted 100 ms with 100 ms interval at −50 mV between the depolarizing stimuli, was conceivably reduced to 1.78 ± 0.07 s (n = 8, p < 0.05, Figure 9(Ba)). In the presence of 300 µM PHB, the decaying time constant in response to 10 s repetitive depolarizing pulses was obviously decreased to 0.92 ± 0.05 s (n = 8, p < 0.05, Figure 9(Bb)). This decrease in the decaying time constant mediated by PHB could not be modified by FLM (10 µM). Thus, it is possible that the excessive accumulative inactivation of I_K(DR) during the exposure to PHB might be irrelevant to GABA_A receptors.

4. Discussion

The noticeable findings demonstrated in this work are that PHB can regulate the magnitude of multiple types of ionic currents (i.e., I_Na, I_K(erg), I_K(M), and I_K(DR)) residing in Neuro-2a cells. Consistent with previous observations [54], the presence of PHB itself suppressed peak I_Na in a concentration-dependent manner with an estimated IC₅₀ of 83 µM (Figure 1). However, the magnitude of PHB-mediated block in these ionic currents failed to be overcome by the subsequent addition of FLM or chlorotoxin. The cumulative inhibition of ionic currents activated during pulse train stimulation was facilitated by PHB. The PHB-induced rise in I_Na decay during repetitive stimulation was reversed by Tef. These experimental observations reflected that the reduction of I_Na, I_K(erg), I_K(M), and I_K(DR) caused by PHB in Neuro-2a cells tends to be acute and may be independent of GABA_A receptor-mediated chloride currents. In addition, these actions could potentially contribute to PHB-induced perturbations on the electrical behaviors of excitable cells (e.g., the discharge patterns in the high-frequency firing of action potentials [APs]), presuming that similar in vivo findings appear [55,56].

Previous work has demonstrated that the train of depolarizing pulses could be efficacious in perturbing the magnitude of I_Na, i.e., current decaying over time in an exponential fashion [14]. This blocking effect is essential in regulating sensory transduction because the driven spike rates can reach hundreds of hertz in response to strong stimuli. As a corollary, the reduced availability of Na_V channels during prolonged repetitive activity (i.e., long-lasting trains of high-frequency APs) would attenuate the spike waveform, and Ca²⁺ flux and Ca²⁺-dependent exocytosis would be subsequently impaired at the nerve terminal. In our study, PHB was noted to shorten the τ value of I_Na decay responding to PT stimulation (Figure 2). Thus, it would further diminish Na_V-channel availability at the same time.

The magnitude of I_Na could be rapidly declined in an exponential manner during high-frequency stimulation, as demonstrated previously [14,40,42] (Figure 2). In other words, during prolonged repetitive activity, the availability of Na_V channels was profoundly decreased in a time-dependent fashion. By comparison, the decaying rate of I_Na during such high PT stimulation was noticeably faster than that of I_K(erg), I_K(M), or I_K(DR) (Figure 4, Figure 7, and Figure 9) seen in Neuro-2a cells. Although the gating kinetics or inactivation-dependent mechanisms residing in these K⁺ currents are overly distinguishable [21,24,31,46,52], the difference in current decaying during PT stimulation could be of particular significance. The main reason for this is because the magnitude of these K⁺ currents (i.e., resurgent K⁺ tail currents) could maintain the membrane in a spike-ready state, thus conferring optimal repriming of I_Na during the occurrence of prolonged repetitive activity [27,31]. In this scenario, the augmentation of cumulative I_K(erg), I_K(M), or I_K(DR) inactivation caused by PHB during high-frequency activity is relevant because it is able to suppress the rapid firing of neuronal APs and cause an additional loss-of-change in stable waveform and high-fidelity synaptic signaling, particularly in high-frequency firing [27]. Therefore, during high-frequency firing of neurons, the presence of PHB itself would decrease the magnitude of post-spike and steady-state currents leading to diminishing the subthreshold depolarized potential.

In the current study, PHB could slow the recovery of the I_K(erg) block and increase the decay of I_K(erg) activated by the envelope-of-tail method (Figure 5). These findings suggested that the molecule of PHB is capable of interacting with the open states (conformations) of the K_erg channels present in Neuro-2a cells.

Distinguishable from a recent study [28], the I_K(M) present in Neuro-2a cells was directly suppressed by PHB. However, it should be emphasized that the I_K(M) amplitude can gradually and progressively arise during repetitive APs because of its slow activation and deactivation kinetics [27]. Upon high-frequency stimulation, the accumulation of I_K(M) is allowed to hyperpolarize the after-potential and consequently speed up the recovery of Na_V channels from inactivation as well to increase the availability of these channels. The PHB facilitated the cumulative inhibition of I_K(M) during PT stimulation (Figure 7). It means that the waveform of neuronal action potentials (APs) in high frequency could be distorted by PHB, and presynaptic signaling occurring across the full dynamic range of the system was thereafter potentially perturbed.

Despite its rapid deactivation kinetics, I_K(DR) amplitude (e.g., K_V3.1-encoded current), identified previously as a timely resurgent K⁺ current, can be progressively raised during high-frequency stimulation [31]. During high-frequency stimulation, the accumulation of I_K(DR) can be allowed to hyperpolarize the after-potential as well as speed up the recovery of Na_V channels from inactivation. In this study, we found that the I_K(DR) in Neuro-2a cells can be susceptible to being blocked by PHB, and the cumulative inhibition of I_K(DR) during prolonged (i.e., 10 s) repetitive stimulation was enhanced (Figure 9). Consequently, the magnitude of I_Na recovery from current inactivation during PT stimulation would become slowed owing to the enhanced cumulative inhibition of I_K(erg), I_K(DR), and I_K(M) [27,31,57]. In this scenario, as cells were exposed to PHB, the high-frequency firing of neuronal APs would lose the emergence of APs’ stable waveforms leading to serious perturbations in high-fidelity synaptic signaling (e.g., the presynaptic release of neurotransmitters) [56]. In other words, the negative feedback mechanism on K_V channel closure during high PT stimulation would be impaired during the exposure to PHB [27,31]. Therefore, susceptibility to PHB in vivo may depend on the pre-existing resting potential level, the high-frequency firing, and the concentration of PHB.

From previous pharmacokinetic studies, the recommended plasma concentrations of PHB for anti-seizure activities ranged between 10 and 35 µg/mL (or 43 and 151 µM), while for prophylaxis against febrile convulsions was around 15 µg/mM (or 65 µM) [3,5,58]. Because PHB is a lipophilic compound, its membrane concentration would be higher than blood levels. This study showed that the PHB concentration for the half-maximal inhibition of I_Na seen in Neuro-2a cells was 83 µM, a value within the clinically applied doses. The observed effects of PHB presented herein can achieve clinical concentration, especially for the treatment of seizure activities lined to high-frequency AP firing.

By synergistic inhibition of multiple ionic currents, including I_Na, I_K(erg), I_K(M), or I_K(DR) in a frequency-dependent manner, PHB might induce other unpredictable electrophysiological responses, in addition to solely being an activator of GABA_A receptors. The clinical use of PHB should take these effects into account. However, the findings in the current study are limited to Neuro-2a cells. Further studies will have to validate these in other excitable cells or in vivo.