The Integrated Effects of Brivaracetam, a Selective Analog of Levetiracetam, on Ionic Currents and Neuronal Excitability

biomedicines

Article The Integrated Effects of Brivaracetam, a Selective Analog of Levetiracetam, on Ionic Currents and Neuronal Excitability

Te-Yu Hung 1, Sheng-Nan Wu 2,* and Chin-Wei Huang 3,*

1 Department of Pediatrics, Chi-Mei Medical Center, Tainan 71004, Taiwan; [email protected] 2 Department of Physiology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan 3 Department of Neurology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan * Correspondence: [email protected] (S.-N.W.); [email protected] (C.-W.H.)

Abstract: Brivaracetam (BRV) is recognized as a novel third-generation antiepileptic drug approved for the treatment of epilepsy. Emerging evidence has demonstrated that it has potentially better efficacy and tolerability than its analog, Levetiracetam (LEV). This, however, cannot be explained by their common synaptic vesicle-binding mechanism. Whether BRV can affect different ionic currents and concert these effects to alter neuronal excitability remains unclear. With the aid of patch clamp technology, we found that BRV concentration dependently inhibited the depolarization- + + induced M-type K current (IK(M)), decreased the delayed-rectifier K current (IK(DR)), and decreased the hyperpolarization-activated cation current in GH3 neurons. However, it had a concentration- + dependent inhibition on voltage-gated Na current (INa). Under an inside-out patch configuration, 2+ + a bath application of BRV increased the open probability of large-conductance Ca -activated K channels. Furthermore, in mHippoE-14 hippocampal neurons, the whole-cell INa was effectively

Citation: Hung, T.-Y.; Wu, S.-N.; depressed by BRV. In simulated modeling of hippocampal neurons, BRV was observed to reduce Huang, C.-W. The Integrated Effects the ﬁring of the action potentials (APs) concurrently with decreases in the AP amplitude. In animal of Brivaracetam, a Selective Analog of models, BRV ameliorated acute seizures in both OD-1 and lithium-pilocarpine epilepsy models. Levetiracetam, on Ionic Currents and However, LEV had effects in the latter only. Collectively, our study demonstrated BRV’s multiple Neuronal Excitability. Biomedicines ionic mechanism in electrically excitable cells and a potential concerted effect on neuronal excitability 2021, 9, 369. https://doi.org/ and hyperexcitability disorders. 10.3390/biomedicines9040369 Keywords: brivaracetam; M-type K+ current; voltage-gated Na+ current; large-conductance Ca2+- Academic Editor: Amirata activated K+ channel; neuron; seizure Saei Dibavar

Received: 14 February 2021 Accepted: 26 March 2021 1. Introduction Published: 1 April 2021 ® ® Brivaracetam (BRV; Brivact , Brivlera , UCB34714, C11H20N2O2), a chemical analog

Publisher’s Note: MDPI stays neutral of levetiracetam (LEV), is an orally or intravenously bioavailable racetam derivative with with regard to jurisdictional claims in anticonvulsant (antiepileptic) properties that has appeared in a growing number of research published maps and institutional affil- papers [1–33]. Of note, it has also been recognized to be efficacious in the treatment of iations. epilepsy and status epilepticus [9,17,30,34–38]. BRV has also been reported to attenuate pain behavior in a murine model of neuropathic pain [2,39]. BRV was also previously observed to exert anti-neoplastic effects in glioma cells [40]. It has been demonstrated that BRV can interfere with the functional activities of neurons (e.g., hippocampal neurons) or endocrine cells (e.g., pituitary lac- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. totrophs) by binding with high affinity to the synaptic or endocrine vesicle protein 2A This article is an open access article (SV2A) [9,15,39,41–45]. SV2A has been recognized as an important broad marker for neu- distributed under the terms and roendocrine cells [41]. It has been reported to be 10–30-fold more potent than LEV, with conditions of the Creative Commons high efficacy in a wide range of experimental models of focal and generalized seizures. Attribution (CC BY) license (https:// As a potential medication with significantly high SV2A affinity, it is thus important to creativecommons.org/licenses/by/ investigate its mechanism on neuroendocrine cells and hippocampal neurons. Furthermore, 4.0/). BRV has been demonstrated to have higher potency and efficacy than LEV in experimental

Biomedicines 2021, 9, 369. https://doi.org/10.3390/biomedicines9040369 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 369 2 of 19

models of epilepsy [46]. However, to the best of our knowledge, there is little information available with respect to the effects of BRV on different types of membrane ionic currents in excitatory endocrine cells or neurons or on neuronal excitability, although a previous study showed the ability of BRV to alter the magnitude of voltage-gated Na+ current or M-type K+ current residing in neurons [42,47]. Furthermore, clinical reports have demonstrated that BRV may be a useful treatment option in patients who have previously failed to respond to or tolerate LEV [48], and it has been reported that BRV has better clinical efﬁcacy and tolerability than LEV [26,28] that cannot be explained simply by the common synaptic vesicle binding property mechanism. In light of these considerations, we attempted to characterize the effects of BRV on + + membrane ionic currents (e.g., M-type K current (IK(M)), a delayed-rectiﬁer K current + (IK(DR)), hyperpolarization-activated cation current (Ih), a voltage-gated Na current (INa), 2+ + and a large-conductance Ca -activated K (BKCa) channel) existing in neuroendocrine and hippocampal neurons in order to investigate the integrated effects on neuronal excitability and hyperexcitability using simulation modeling and different animal models of epilepsy.

2. Materials and Methods 2.1. Chemicals, Drugs, and Solutions Used in This Study Brivaracetam (BRV,Brivact®, Brivlera®, UCB34714, ((2S)-2-[(4R)-2-oxo-4-propylpyrrolidin- 1-yl]butanamide, C11H20N2O2, https://pubchem.ncbi.nlm.nih.gov/compound/Brivaracetam, accessed on 25 March 2021) and LEV (Keppra®, (S)-2(2-oxopyrrolidin-1-yl)butanamide) were kindly provided by UCB (Union Chimique Belge) Pharma (PRA Health Sciences, Taipei, Taiwan). GAL-021 and PF1022A were acquired from MedChemExpress (Everything Biotech, New Taipei City, Taiwan); cilobradine was obtained from Cayman (Excel Biomedi- cal, Taipei, Taiwan); 2-chloro-α, α-diphenylbenzeneacetonitrile (TRAM39) was obtained from Tocris (Union Biomed Inc., Taipei, Taiwan), and tefluthrhin, tetraethylammonium chloride and tetrodotoxin were obtained from Sigma-Aldrich (Merck Ltd., Taipei, Taiwan). Unless specified otherwise, culture media, fetal bovine serum, horse serum, L-glutamine, penicillin-streptomycin and trypsin/EDTA were obtained from HyCloneTM (Thermo Fisher Scientific; Level Biotech, Tainan, Taiwan), while all other chemicals, including CdCl2, CsCl, CsOH, EGTA and HEPES, were of laboratory grade and obtained from standard sources. We used twice-distilled water that had been deionized through a Millipore-Q system (Merck, Ltd., Taipei, Taiwan) in all experiments. The ionic composition of the bath solution (i.e., HEPES-buffered normal Tyrode’s solution) was: NaCl 136.5 mM, KCl 5.4 mM, CaCl2 1.8 mM, MgCl2 0.53 mM, glucose 5.5 mM, and HEPES 5.5 mM adjusted to pH 7.4 with NaOH. To measure the macroscopic + K currents (e.g., IK(M) or IK(DR)), we filled the recording electrode with a solution: K- aspartate 130 mM, KCl 20 mM, MgCl2 1 mM, Na2ATP 3 mM, Na2GTP 0.1 mM, EGTA 0.1 mM, and HEPES 5 mM adjusted to pH 7.2 with KOH. To study the BKCa-channel activity measured under an inside-out configuration, the bath solution contained a high + K solution: KCl 130 mM, NaCl 10 mM, MgCl2 3 mM, glucose 6 mM, and HEPES 10 mM titrated to 7.4 with KOH, while the pipette solution contained KCl 145 mM, MgCl2 2 mM, and HEPES 5 mM titrated to 7.4 with KOH. The value of the free Ca2+ concentration was estimated in this study, assuming that there was a dissociation constant of 0.1 µM for EGTA and Ca2+ (at pH 7.2). For example, to provide 0.1 µM Ca2+ in the bath solution, 1 mM EGTA and 0.5 mM CaCl2 were added. In this study, we commonly filtered the pipette solutions and culture media with an Acrodisc® syringe filter with a 0.2 µm Super® membrane (Bio-Check Lab., Pall Corp., Taipei, Taiwan).

2.2. Cell Culture

The pituitary adenomatous cell line, GH3, was acquired from the Bioresource Col- lection and Research Center (BCRC-60015, http://catalog.bcrc.ﬁrdi.org.tw/BcrcContent? bid=60015 (accessed on 26 March 2021), Hsinchu, Taiwan) [49], and the embryonic mouse hippocampal cell line, mHippoE-14, was obtained from Cedarlane CELLutions Biosystems Biomedicines 2021, 9, 369 3 of 19

Inc. (Hycell International Co., Taipei, Taiwan) [50,51]. The GH3 cells were maintained in Ham’s F-12 medium supplemented with 2.5% fetal bovine serum (v/v), 15% horse serum (v/v), and 2 mM L-glutamine, and the mHippoE-14 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (v/v) and 2 mM L-glutamine. The cells were grown as a monolayer culture in a humidified environment of ◦ 5% CO2/95% air at 37 C. In order to be well differentiated, the GH3 cells were transferred to serum- and Ca2+-free mediums. Electrical recordings were performed 5 or 6 days after the cells had been cultured to 60–80% confluence.

2.3. Electrophysiological Measurements On the day of the experiments, the cells were dispersed with a 1% trypsin/EDTA solution, and a few drops of cell suspension were quickly transferred to a custom-built recording chamber mounted on the stage of an inverted DM-II microscope (Leica; Major Instruments, Kaohsiung, Taiwan). They were immersed at room temperature (20–25 ◦C) in normal Tyrode’s solution, the composition of which was provided above. We recorded different types of ionic currents in the whole-cell, cell-attached, or inside-out mode of a standard patch-clamp technique with dynamic adaptive suction (i.e., a decremental change in the suction pressure in response to a progressive increase in the seal resistance), with the aid of either an Axoclamp-2B (Molecular Devices, Sunnyvale, CA, USA) or an RK-400 ampliﬁer (Bio-Logic, Claix, France). Consistent with previous observations [52], the formation of a bleb of membrane lipid in the electrode tip based on microscopic observation of giga-Ω seal formation was observed in our study. The microelectrodes used were prepared from Kimax-51 borosilicate capillaries with a 1.5 mm outer diameter (#34500; Kimble; Dogger, New Taipei City, Taiwan) by using a PP-83 vertical puller (Narishige; Taiwan Instrument, Taipei, Taiwan). Their tip resistance ranged between 3 and 5 MΩ, and they were ﬁlled with the various internal solutions described above. During the measurements, the recorded area on the vibration-free table was shielded using a Faraday cage (Scitech, Seoul, Korea). The liquid–liquid junction potential, which commonly appears when the composition of the pipette solution is different from that in the bath, was corrected before the seal formation. The main rationale for the concentrations applied is to use the level which could be clinically achievable and of therapeutic relevance.

2.4. Data Recordings The signals, comprising potential and current tracings, were monitored on an HM- 507 oscilloscope (Hameg, East Meadow, NY, USA) and digitally stored online at 10 kHz in an ASUS VivoBook Flip 14 laptop computer (TP412FA-0131A10210U; ASUS, Tainan, Taiwan) equipped with a 12-bit resolution Digidata 1440A interface (Molecular Devices). During the measurements with either analog-to-digital or digital-to-analog conversion, the latter device was controlled using pCLAMP v.10.7 software (Molecular Devices) run on Microsoft Windows 10 (Redmond, WA, USA). A laptop computer was put on the top of an adjustable Cookskin stand (Ningbo, Zheijiang, China) to allow efﬁcient manipulation during the recordings.

2.5. Data Analyses

To assess the percentage inhibition of BRV on the IK(M) amplitude, each cell was voltage-clamped at −50 mV, and a 1-sec depolarizing pulse to −10 mV was applied. The examined cell was briefly depolarized from −80 to −10 mV. The IK(M) and INa amplitudes measured at the beginning or end of the depolarizing pulses in the presence of various concentrations of BRV were compared with the control value (i.e., BRV was not present). The concentration-response data for BRV-induced inhibition of IK(M) or peak INa were well fitted with the modified Hill equation (i.e., the 3-parameter logistic equation):

E × (BRV)nH Percentage inhibition (%) = max (1) nH nH IC50 + (BRV) Biomedicines 2021, 9, 369 4 of 19

where (BRV) represents the BRV concentration; IC50 and nH are the concentrations required for a 50% inhibition and Hill coefficient, respectively, and Emax represents the BRV-mediated maximal block of IK(M) or the peak INa. To assess the steady-state inactivation curve of INa measured with or without the addition of BRV, we employed a two-step voltage profile. The relationship between the normalized amplitude of peak INa and the different conditioning potentials was least- squares fitted to a Boltzmann function in the following form:

I 1 = n ( − ) o (2) Imax V V1/2 qF 1 + exp RT

where V is the conditioning potential; V1/2 the potential at which a half-maximal decrease occurs; Imax is the maximal amplitude of INa; q is the apparent gating charge in the inactivation curve of the current (i.e., the charge across the membrane’s electrical ﬁeld between the closed and open conformations); F is the Faraday constant; R is the universal gas constant; T is the absolute temperature, and RT/F = 25.2 mV.

2.6. Single-Channel Analyses

The unitary current events of the digitized BKCa channels were assessed using a pCLAMP v.10.7 (Molecular Devices). Multi-Gaussian adjustments of the amplitude dis- tributions occurring among the channel events were applied to determine the opening channel event. The functional independence among the channels was validated as the observed stationary probabilities were compared. The open-state probabilities were evaluated using an iterative process to minimize the X2 value calculated from a sufﬁcient number of independent observations.

2.7. Simulation Modeling To evaluate how BRV could adjust the ﬁring of action potentials (APs), a theoretical model was adapted from a previous work [53]. The model is based largely on the biophysi- cal properties of hippocampal CA1 pyramidal neurons and consists of the delayed-rectiﬁer K+ current, the M-type K+ current, the transient K+ current, the Ca2+-activated K+ current, the Na+ current, and the Ca2+ current. A hyperpolarization-activated cation current adapted from a previous work [54] was also included in the model. The conductance values used to solve the set of differential equations are listed in Table1.

Table 1. Parametric values used for the modeling of hippocampal CA1 pyramidal neurons in an attempt to mimic the experimental results obtained in the control, where brivaracetam (BRV) was not present and during exposure to 10 µM BRV.

Symbol Description Value (in Control) Value (in the Presence of 10 µM BRV) Cm Membrane capacitance (pF) 1 1 + 2 gNa Na current conductance (mS/cm ) 35 17.5 2+ 2 gCa Ca current conductance (mS/cm ) 0.08 0.08 + 2 gKDR Delayed-rectiﬁer K current conductance (mS/cm ) 6.0 5.4 + 2 gA A-type K current conductance (mS/cm ) 1.4 1.4 + 2 gM M-type K current conductance (mS/cm ) 1 0.7 2+ + 2 gKCa Ca -activated K current conductance (mS/cm ) 10 20 Hyperpolarization-activated cation current g 0.4 0.36 h conductance (mS/cm2)

2.8. Animal Experiments All experiments, including the animal procedures, were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) (Approval No: 110147, Date: 20 February 2021) at National Cheng Kung University, Tainan, Taiwan. Adult Sprague-Dawley male rats weighing 180 to 200 g were purchased from National Cheng Kung University. They were housed in the university’s Animal Center and allowed free access to water and a Biomedicines 2021, 9, 369 5 of 19

pelleted rodent diet (Richmond Standard; PMI Feeds, St Louis, MO, USA). All efforts were made to minimize the number of rats used. The animals were divided into two groups (OD-1 and lithium-pilocarpine). We previously characterized the OD-1 group and its effect as a novel animal model [54]. The OD-1 group was stereotactically injected with 5 ng OD-1 (12 mouse LD50) in the hippocampus (coordinates 4.1 mm caudal, 3.9 mm lateral to the bregma and 3.8 mm below the cortical surface) under general anesthesia (zoletil, 0.1 mL/100 g, intraperitoneal injection (ip)) and analgesia (nalbuphine, 5 mg/kg, ip). After recovery from anesthesia (24 h after injection), the OD-1 group was then divided into three groups given normal saline (NS, 0.1 mL/100g, an intraperitoneal injection (ip)), LEV (500 mg/kg, ip), or BRV (100 mg/kg, ip). The acute seizures were carefully monitored and scored in the following eight hours. The rats in the pilocaprine group were divided into three groups given NS (0.1 mL/100 g, ip), LEV (500 mg/kg, ip), or BRV (100 mg/kg, ip) 20 min before the pilocarpine injection. The lithium-pilocarpine group was injected with lithium chloride (3 meq/kg; ip) and methylscopolamine (25 mg/kg; subcutaneous (sc)) before they were subjected to pilocarpine (60 mg/kg; subcutaneous injection (sc))-induced seizures. In both of the OD-1 and lithium-pilocarpine groups, the seizure characteristics of the rats during acute seizures were similar to those reported elsewhere [51,55,56]. The seizures were scored using the Racine scale [56]. The rats were given zoletil (50 mg/kg, ip), xylazine (20 mg, ip), and atropine (0.2 mg/kg, sc) to diminish the seizures if their status epilepticus lasted for 20 min [51,55–58]. Mortality was calculated during the ﬁrst 24 h after seizure onset. All rats were continuously monitored for the ﬁrst 24 h after they achieved status epilepticus by two experienced research assistants. The rats were given supportive care: body temperature maintenance with a resistive heating system, food, and adequate hydration with normal saline (0.9% w/v of NaCl, 308 mOsm/L). Any animals showing intense signs of acute respiratory distress were immediately euthanized by overdosing with sodium pentobarbital (150 mg/kg, ip). In OD-1 model, we evaluated the parameter severe seizures as an indicator of higher neuronal excitotoxicity. In pilocarpine model, the latency to acute stage 3 seizures, number of rats with severe seizures and acute mortality were used to evaluate the neuronal excitotoxicity.

2.9. Statistical Analyses Linear or nonlinear curve-fitting (e.g., exponential curve or sigmoidal Hill and Boltz- mann equations) to any given data sets was undertaken, with the goodness of fit assessed using either Microsoft Excel® v.2016 (Redmond, WA, USA) or OriginPro v.2016 (OriginLab; Schmidt, Taipei, Taiwan). Values are provided as means ± SEM with sample sizes (n), which indicate the number of GH3 cells or mHippoE-14 neurons from which the experimental data were collected. The Student’s t-test and a one-way analysis of variance (ANOVA) followed by post-hoc Fisher’s least-significant difference test for multiple comparisons were performed. However, the data were examined using the nonparametric Kruskal–Wallis test, subject to possible violations in the normality underlying the ANOVA. Differences were considered statistically significant when the p-value was less than 0.05.

3. Results + 3.1. Inhibitory Effect of BRV on the Amplitude of M-Type K Current (IK(M)) The initial stage of the experiments was undertaken to evaluate the effect of BRV + 2+ on the IK(M) inherently existing in the GH3 cells. We bathed the cells in a high-K , Ca - free solution, and, during the recordings, we backﬁlled the pipette using a K+-containing solution comprising: K-aspartate 130 mM, KCl 20 mM, MgCl2 1 mM, Na2ATP 3 mM, Na2GTP 0.1 mM, EGTA 0.1 mM, and HEPES 5 mM adjusted to pH 7.2 with KOH. When the whole-cell mode was ﬁrmly established, we voltage-clamped the cell at −50 mV and thereafter applied a 1 s depolarizing pulse ranging from −50 to −10 mV. As expected, an inward current with a slowly activating time course was evoked. This current has Biomedicines 2021, 9, x FOR PEER REVIEW 6 of 19

ANOVA. Differences were considered statistically significant when the p-value was less than 0.05.

3. Results

3.1. Inhibitory Effect of BRV on the Amplitude of M-Type K+ Current (IK(M)) The initial stage of the experiments was undertaken to evaluate the effect of BRV on the IK(M) inherently existing in the GH3 cells. We bathed the cells in a high-K+, Ca2+-free solution, and, during the recordings, we backfilled the pipette using a K+-containing solution comprising: K-aspartate 130 mM, KCl 20 mM, MgCl2 1 mM, Na2ATP 3 mM, Na2GTP Biomedicines 2021, 9, 369 0.1 mM, EGTA 0.1 mM, and HEPES 5 mM adjusted to pH 7.2 with KOH. When the whole-6 of 19 cell mode was firmly established, we voltage-clamped the cell at −50 mV and thereafter applied a 1 s depolarizing pulse ranging from −50 to −10 mV. As expected, an inward current with a slowly activating time course was evoked. This current has previously been identifiedpreviously to been be identiﬁedIK(M) [59–61]. tobe It IwasK(M) clearly[59–61]. observed It was clearly that, observedas the cells that, were as the exposed cells were to differentexposed toconcentrations different concentrations of BRV, the of amplitude BRV, the amplitude of IK(M) progressively of IK(M) progressively decreased decreased (Figure µ I 1A).(Figure For1 A).example, For example, the addition the addition of 10 µM of BRV 10 decreasedM BRV decreased the IK(M) theamplitudeK(M) amplitude from 78 ± from 8 to 4278 ±± 48 pA to 42(n ±= 8,4 pAp < 0.05). (n = 8, Afterp < 0.05). it was After removed, it was removed,the current the amplitude current amplitude returned returnedto 74 ± 6 µ pAto 74(n ±= 7,6 pAp < 0.05). (n = 7, Thep < activation 0.05). The time activation course time in the course presence in the of presenceBRV (10 µM) of BRV slowed, (10 M)as slowed, as evidenced by a signiﬁcant prolongation in the activation time constant (τ ) evidenced by a significant prolongation in the activation time constant (τact) of the currentact ± ± fromof the 92 current ± 11 to 122 from ± 14 92 msec11 (n to = 8, 122 p < 0.05).14 msecMoreover, (n = 8,whenp < the 0.05). differences Moreover, in the when current the µ tracesdifferences between in thethe currentabsence traces and presence between of the 10 absenceor 30 µM and BRV presence were taken, of 10 the or 30net changeM BRV were taken, the net change in the membrane currents, i.e., the BRV-sensitive component, in the membrane currents, i.e., the BRV-sensitive component, was obtained (Figure 1B). was obtained (Figure1B). These BRV-sensitive inward currents exhibited time-dependent These BRV-sensitive inward currents exhibited time-dependent activation and deactiva- activation and deactivation. Figure1C shows that cell exposure to BRV can result in a tion. Figure 1C shows that cell exposure to BRV can result in a concentration-dependent concentration-dependent decrease in the amplitude of IK(M) elicited in response to 1 s decrease in the amplitude of IK(M) elicited in response to 1 s step depolarization. The IC50 step depolarization. The IC50 value needed for BRV-perturbed inhibition of IK(M) was value needed for BRV-perturbed inhibition of IK(M) was estimated to be 6.5 µM. Therefore, estimated to be 6.5 µM. Therefore, BRV is capable of producing a depressant action on BRV is capable of producing a depressant action on depolarization-induced IK(M). depolarization-induced IK(M). A B

-20 0 ab mV -40 -50 0 500 1000 1500 2000 pA msec

a: BRV-sensitive current (10 μM) 0 -100 b: BRV-sensitive current (30 μM) a b c d 0 500 1000 1500 2000 -50 100 C msec

pA 80

60 -100 a: control b: 10 μM BRV 40 c: 30 μM BRV d: washout 20 -150 0 500 1000 1500 2000 (%) inhibition Percentage 0 msec 0.1 1 10 100 [BRV], μM

+ Figure 1. Inhibitory effect of BRV on M-type K current (IK(M)) in GH3 cells. In these experiments, + 2+ Figurecells were 1. Inhibitory immersed effect in high-K of BRV ,on Ca M-type-free K solution,+ current and (IK(M) the) in recording GH3 cells. electrode In these experiments, was ﬁlled with + + 2+ cellsthe Kwere-containing immersed solution in high-K comprising:, Ca -free K-aspartatesolution, and 130 the mM, recording KCl 20 electrode mM, MgCl was2 1filled mM, with Na2 theATP + K3-containing mM, Na2GTP solution 0.1 mM, comprising: EGTA 0.1 K-aspartate mM, and 130 HEPES mM, 5KCl mM 20 adjusted mM, MgCl to2 pH1 mM, 7.2 Na with2ATP KOH; 3 mM, (A) Na2GTP 0.1 mM, EGTA 0.1 mM, and HEPES 5 mM adjusted to pH 7.2 with KOH; (A) Representa- Representative IK(M) traces evoked by a 1 s depolarizing pulse from −50 to −10 mV (indicated in tivethe upperIK(M) traces part evoked of the ﬁgure).by a 1 s (a):depolarizing control (i.e., pulse BRV from was −50 not to present);−10 mV (indicated (b): 10 µM in BRV; the upper (c): 30 partµM of the figure). (a): control (i.e., BRV was not present); (b): 10 µM BRV; (c): 30 µM BRV; (d): washout BRV; (d): washout of BRV. It should be noted that the trajectories of the deactivating currents were of BRV. It should be noted that the trajectories of the deactivating currents were partly cut off for partly cut off for the purpose of illustration; (B) BRV-sensitive inward current (a–b (green trace) or a–c (red trace) in panel (A)) (a): BRV-sensitive current at 10 µM BRV; (b): BRV-sensitive current at 30

µM BRV; (C) Concentration-dependent inhibition of BRV on IK(M) amplitude measured from GH3 cells (mean ± SEM; n = 8). Current amplitude obtained in the absence and presence of different BRV concentrations (0.3–100 µM) was taken at the endpoint of depolarization pulses ranging from −50 to −10 mV. A smooth, continuous line was obtained by ﬁtting the experimental results to the modiﬁed Hill equation (as elaborated in the Materials and Methods section). The vertical broken line points

out the IC50 value (i.e., 6.5 µM) needed for BRV-mediated inhibition of IK(M) in these cells. + 3.2. Mild Inhibition of BRV on Delayed-Rectiﬁer K Current (IK(DR)) + The question as to whether BRV affects other types of K currents (e.g., IK(DR)) was raised. Previous reports have shown the ability of LEV to modify the amplitude and gating of IK(DR) [62]. The subsequent experiments were therefore performed to evaluate whether IK(DR) could be modiﬁed in the presence of BRV. To elicit a family of IK(DR) (Figure2A ), cells Biomedicines 2021, 9, x FOR PEER REVIEW 7 of 19

the purpose of illustration; (B) BRV-sensitive inward current (a–b (green trace) or a–c (red trace) in panel (A)) (a): BRV-sensitive current at 10 µM BRV; (b): BRV-sensitive current at 30 µM BRV; (C) Concentration-dependent inhibition of BRV on IK(M) amplitude measured from GH3 cells (mean ± SEM; n = 8). Current amplitude obtained in the absence and presence of different BRV concentrations (0.3–100 µM) was taken at the endpoint of depolarization pulses ranging from −50 to −10 mV. A smooth, continuous line was obtained by fitting the experimental results to the modified Hill equation (as elaborated in the Materials and Methods section). The vertical broken line points out the IC50 value (i.e., 6.5 µM) needed for BRV-mediated inhibition of IK(M) in these cells.

3.2. Mild Inhibition of BRV on Delayed-Rectifier K+ Current (IK(DR))

The question as to whether BRV affects other types of K+ currents (e.g., IK(DR)) was Biomedicines 2021 9 , , 369 raised. Previous reports have shown the ability of LEV to modify the amplitude7 of 19 and gating of IK(DR) [62]. The subsequent experiments were therefore performed to evaluate whether IK(DR) could be modified in the presence of BRV. To elicit a family of IK(DR) (Figure were immersed2A), cells in Ca were2+-free immersed Tyrode’s in solutionCa2+-free containingTyrode’s solution 1 µM tetrodotoxin containing 1 and µM 0.5 tetrodotoxin mM and + + CdCl2, and0.5 the mM recording CdCl2, and electrode the recording was filled electrode with Kwas-containing filled with solution.K -containing During solution. the During measurements,the measurements, the examined the cell examined was maintained cell was atmaintained−50 mV, andat −50 various mV, and voltage various steps voltage steps ranging betweenranging− between60 and +50 −60 mV and at +50 intervals mV at ofintervals 10 mV were of 10 applied mV were to applied evoke IK(DR) to evoke[61,62 IK(DR)]. [61,62]. Under theseUnder conditions, these conditions, BRV at a concentrationBRV at a concentration of 3 µM was of 3 not µM found was not to have found any to effecthave any effect on IK(DR),on as measuredIK(DR), as measured throughout throughout the entire the voltage-clamp entire voltage-clamp step. For example,step. For asexample, the cells as the cells were depolarizedwere depolarized from −50 from to +50 −50 mV, to the+50 ImV,K(DR) theamplitude IK(DR) amplitude in the absence in the absence and presence and presence of of 3 µM BRV3 µM did BRV not did differ not differ significantly significantly (822 ±(82242 ± pA 42 pA (control) (control) vs. vs. 821 821± ±22 22 pA pA (the(the presence presence ofof BRV);BRV); nn == 8, p > 0.05). 0.05). However, However, when when the the cells cells were were exposed exposed to to 10 10 µMµM BRV, BRV, the ampli- the amplitudetude ofof IIK(DR)K(DR) slightly,slightly, but significantly, significantly, decreaseddecreased (Figure(Figure2 A,B).2A,B). For For example, example, at at +50 mV, +50 mV, thethe current current amplitude amplitude significantly significantly declined declined from from 834 834± ±49 49 to to 711 711± ±38 38 pA pA ( n(n= = 8 8,, p < 0.05) p < 0.05) duringduring cell cell exposure exposure to to 10 10µM µM BRV. BRV. The Th current-voltagee current-voltage (I-V) relationship(I-V) relationship to IK(DR) to IK(DR) col- collected withlected or with without or without the addition the addition of 10 µM of BRV 10 µM is illustrated BRV is illustrated in Figure 2inB. Figure It could 2B. be It could be observed that,observed unlike that,IK(M) unlike, IK(DR) IK(M)tends, IK(DR) to tends be less tosubject be less tosubject being to altered being byaltered BRV. by BRV.

+ + Figure 2. Mild inhibitionFigure 2. ofMild BRV inhibitionon delayed-rectifier of BRV on K delayed-rectifier current (IK(DR)) in K GHcurrent3 cells.( IInK(DR) these) in experiments, GH3 cells. Inthe these cells were bathed in Ca2+-freeexperiments, Tyrode’s solution the cells containing were bathed 1 µM in Catetrodotoxin2+-free Tyrode’s and 0.5 solution mM CdCl containing2, where 1theµM recording tetrodotoxin electrode and was filled with a K+-containing solution comprising: K-aspartate 130 mM, KCl 20 mM,+ MgCl2 1 mM, Na2ATP 3 mM, Na2GTP 0.5 mM CdCl2, where the recording electrode was filled with a K -containing solution comprising: 0.1 mM, EGTA 0.1 mM, and HEPES 5 mM adjusted to pH 7.2 with KOH. (A) Representative IK(DR) traces obtained in the K-aspartate 130 mM, KCl 20 mM, MgCl2 1 mM, Na2ATP 3 mM, Na2GTP 0.1 mM, EGTA 0.1 mM, absence (upper) and presence of 10 µM BRV. The inset in the upper part of the figure indicates the voltage profile used; and HEPES 5 mM adjusted to pH 7.2 with KOH. (A) Representative IK(DR) traces obtained in the (B) Comparison of the mean current-voltage (IV) of IK(DR) without (○) or with the addition (■) of 10 µM BRV (mean ± SEM; µ n = 8). The currentabsence amplitude (upper) was and measured presence at ofthe 10 endM of BRV. each The voltage inset incommand the upper pulse. part It of should the figure be noted indicates thatthe BRV (30 µM) mildly inhibitedvoltage the profile IK(DR) amplitude used; (B)Comparison at +50 mV. of the mean current-voltage (IV) of IK(DR) without ( ) or with the addition () of 10 µM BRV (mean ± SEM; n = 8). The current amplitude was measured# at the end of each voltage command pulse. It should be noted that BRV (30 µM) mildly inhibited the IK(DR) amplitude at +50 mV.

3.3. Mild Inhibitory Effect on Hyperpolarization-Activated Cation Current (Ih) Caused by BRV We further examined whether BRV could produce any modifications on hyperpolarization- 2+ induced Ih. The experiments were conducted in cells bathed in Ca -free Tyrode’s solution containing 1 µM tetrodotoxin, and the recording electrode was ﬁlled with a K+-containing solution. As depicted in Figure3A, the 2 s long hyperpolarizing command voltages ranging from −40 to −120 mV could readily evoke an inward current with slowly activating and deactivating time courses in response to such sustained hyperpolarization. This type of ionic current has been previously identiﬁed to be Ih [49,63,64]. When the cells were exposed to BRV (3 µM), the Ih amplitude was unaffected. However, BRV at a concentration of 10 µM resulted in a lessening in the Ih amplitude from 347 ± 28 to 313 ± 24 pA (n = 7, p < 0.05). Additionally, in the continued presence of 10 µM BRV, subsequent addition of 3 µM cilobradine was observed to lessen the current amplitude further, as demonstrated Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 19

3.3. Mild Inhibitory Effect on Hyperpolarization-Activated Cation Current (Ih) Caused by BRV We further examined whether BRV could produce any modifications on hyperpolarization-induced Ih. The experiments were conducted in cells bathed in Ca2+-free Tyrode’s solution containing 1 µM tetrodotoxin, and the recording electrode was filled with a K+- containing solution. As depicted in Figure 3A, the 2 s long hyperpolarizing command voltages ranging from −40 to −120 mV could readily evoke an inward current with slowly activating and deactivating time courses in response to such sustained hyperpolarization. This type of ionic current has been previously identified to be Ih [49,63,64]. When the cells were exposed to BRV (3 µM), the Ih amplitude was unaffected. However, BRV at a con- Biomedicines 2021, 9, 369 8 of 19 centration of 10 µM resulted in a lessening in the Ih amplitude from 347 ± 28 to 313 ± 24 pA (n = 7, p < 0.05). Additionally, in the continued presence of 10 µM BRV, subsequent addition of 3 µM cilobradine was observed to lessen the current amplitude further, as demonstratedby a signiﬁcant by reductiona significant in current reduction amplitude in current to 129 amplitude± 18 pA to (n 129= 7, ±p <18 0.05) pA ( (Figuren = 7, p3 B).< 0.05) (Figure 3B). Cilobradine was previously used to effectively suppress Ih [65]. Cilobradine was previously used to effectively suppress Ih [65].

Figure 3. Mild inhibition of BRV on hyperpolarization-activated cationic current (Ih) in GH3 cells. FigureThe experiments 3. Mild inhibition were conducted of BRV on inhyperpolar cells immersedization-activated in Ca2+-free cationic Tyrode’s current solution (Ih) containingin GH3 cells. 1 µ M 2+ Thetetrodotoxin, experiments where were theconducted pipette in was cells filled immersed with a in K+ Ca-containing-free Tyrode’s solution solution comprising containing K-aspartate 1 µM tetrodotoxin, where the pipette was filled with a K+-containing solution comprising K-aspartate 130 mM, KCl 20 mM, MgCl2 1 mM, Na2ATP 3 mM, Na2GTP 0.1 mM, EGTA 0.1 mM, and HEPES 130 mM, KCl 20 mM, MgCl2 1 mM, Na2ATP 3 mM, Na2GTP 0.1 mM, EGTA 0.1 mM, and HEPES 5 5 mM adjusted to pH 7.2 with KOH. (A) Representative I traces activated by a 2 s hyperpolarizing mM adjusted to pH 7.2 with KOH. (A) Representative Ih tracesh activated by a 2 s hyperpolarizing voltagevoltage pulse pulse ranging ranging from from −40−40 to to−120−120 mV mV (indicated (indicated in the in the upper upper part part of the of the figure). figure). (1): (1): control, control, (2):(2): 10 10 µMµM BRV, BRV, and and (3): (3): 10 10 µMµM BRV BRV plus 3 µMµM cilobradine (Cil);(Cil); ((BB)) SummarySummary bar bar graph graph showing showing the theeffects effects of of BRV BRV and and BRV BRV plus plus cilobradine cilobradine (Cil) (Cil) in hyperpolarization-activatedin hyperpolarization-activatedIh (mean Ih (mean± SEM; ± SEM;n = n 8). = The8). The current current amplitude amplitude was was measured measured at theat the endpoint endpoint of aof 2 a s 2 hyperpolarizing s hyperpolarizing pulse pulse ranging ranging from from−40 − to40− to120 −120 mV. mV. * indicates * indicates significantly significantly different different from from control control (p < ( 0.05)p < 0.05) and and† indicates † indicates signficantly sign- ficantlydifferent different from BRV from (10 BRV mM) (10 alone mM) group alone (groupp < 0.05). (p < 0.05).

+ 3.4.3.4. Effect Effect of ofBRV BRV on on Voltage-Gated Voltage-Gated Na Na+ CurrentCurrent (INa (I)Na )

InIn the the next next set set of of experiments, experiments, the the INa waswas examined examined in response toto aashort short depolarizing depolariz- ingcommand command voltage voltage to determineto determine whether whether BRV BRV could could modify modify it. Cells it. Cells were were bathed bathed in Ca in2+ - Cafree2+-free Tyrode’s Tyrode’s solution solution containing containing 10 mM 10 mM tetraethylammonium tetraethylammonium chloride, chloride, and and the the electrode elec- + trodewas ﬁlledwas filled with with a Cs a-containing Cs+-containing solution solution comprising: comprising: Cs-aspartate Cs-aspartate 130 130 mM, mM, CsCl CsCl 20 mM, 20 mM,MgCl MgCl2 1 mM,2 1 mM, Na2 ATPNa2ATP 3 mM, 3 mM, Na2GTP Na2 0.1GTP mM, 0.1 EGTAmM, EGTA 0.1 mM, 0.1 and mM, HEPES and HEPES 5 mM adjusted 5 mM adjustedto pH 7.2to pH with 7.2 CsOH. with CsOH. As shown As shown in Figure in Figure4A, when 4A, when the examinedthe examined cell cell was was rapidly rap- − − idlydepolarized depolarized from from80 −80 to to −1010 mV, mV,I NaINa with both a a rapid rapid activation activation and and inactivation inactivation time time course was robustly evoked. As the cells were exposed to BRV, the peak amplitude of INa in response to a brief depolarizing command voltage progressively declined. For example, BRV (10 µM) resulted in an evident reduction in peak INa from 298 ± 19 to 153 ± 11 pA (n = 8, p < 0.05). After the washout of BRV, the current amplitude returned to 291 ± 17 pA (n = 8, p < 0.05). However, neither activation, inactivation, nor deactivation of the time course of INa in response to brief step depolarization was measurably perturbed in the presence of BRV (3 or 10 µM). µ illustrates the mean I-V relationships of peak INa in the absence and presence of 3 or 10 µM BRV. It was observed that the overall I-V relationship to peak INa in these cells was not altered during exposure to BRV, in spite of an obvious lessening in peak INa. The concentration-dependent inhibitory effect of BRV on peak INa amplitude was then determined, as illustrated in Figure4C. According to the modiﬁed Hill equation elaborated in the Materials and Methods Section, the IC50 values for BRV-induced inhibition of peak INa measured at the start of the depolarizing command voltage was Biomedicines 2021, 9, x FOR PEER REVIEW 9 of 19

course was robustly evoked. As the cells were exposed to BRV, the peak amplitude of INa in response to a brief depolarizing command voltage progressively declined. For example, BRV (10 µM) resulted in an evident reduction in peak INa from 298 ± 19 to 153 ± 11 pA (n = 8, p < 0.05). After the washout of BRV, the current amplitude returned to 291 ± 17 pA (n = 8, p < 0.05). However, neither activation, inactivation, nor deactivation of the time course of INa in response to brief step depolarization was measurably perturbed in the presence of BRV (3 or 10 µM). µ illustrates the mean I-V relationships of peak INa in the absence and presence of 3 or 10 µM BRV. It was observed that the overall I-V relationship to peak INa in these cells was not altered during exposure to BRV, in spite of an obvious lessening in peak INa. The concentration-dependent inhibitory effect of BRV on peak INa amplitude was Biomedicines 2021, 9, 369 9 of 19 then determined, as illustrated in Figure 4C. According to the modified Hill equation elaborated in the Materials and Methods Section, the IC50 values for BRV-induced inhibition of peak INa measured at the start of the depolarizing command voltage was found to be 12.2found µM, to beand 12.2 BRVµM, at anda concentration BRV at a concentration of 300 µM almost of 300 µcompletelyM almost completelyeliminate the eliminate current amplitude.the current amplitude.

A B 0 0 -50 mV

0 50 100 -100 msec

pA -200

0 control -300 3 μM BRV 10 μM BRV 0 -100 -80 -60 -40 -20 0 20 40 mV -100 100 C

pA c pA c 80 -200 -200 a: control b 60 b b: 3 μM BRV c: 10 μM BRV d 40 d d: washout -300 a -300 a 015 20 msec

Percentage inhibition (%) inhibition Percentage 0 050100 110100 msec [BRV], μM

+ Figure 4. Inhibitory effects of BRV on voltage-gated Na current (INa) in GH3 cells. The cells were + Figurebathed 4. in Inhibitory Ca2+-free Tyrode’seffects of solutionBRV on voltage-gated containing 10 Na mM current tetraethylammonium (INa) in GH3 cells. chloride, The cells where were the bathed in Ca2+-free Tyrode’s solution containing+ 10 mM tetraethylammonium chloride, where the recording electrode was filled with a Cs -containing solution. (A) Representative INa traces activated recording electrode was filled with a Cs+-containing solution. (A) Representative INa traces acti- by rapid membrane depolarization (indicated in the upper part of the figure). Inset shows an vated by rapid membrane depolarization (indicated in the upper part of the figure). Inset shows expanded record from the dashed box. (a): control (i.e., BRV not present), (b): 3 µM BRV, (c): 10 µM an expanded record from the dashed box. (a): control (i.e., BRV not present), (b): 3 µM BRV, (c): 10 BRV and (d): washout of BRV. (B) Mean I-V relationships of peak I obtained in the absence (•) µM BRV and (d): washout of BRV. (B) Mean I-V relationships of peakNa INa obtained in the absence (and●) and in thein the presence presence of of 3 µ3M µM BRV BRV ( ()○) and and 10 10µ µMM BRVBRV ((4△)) (mean ± SEM;SEM; n n= =7–8). 7–8). The The current current amplitude was measured at the beginning # of each br briefief depolarization. depolarization. It It should should be be noted noted that that cell cell exposure to BRVBRV depresseddepressed thethe amplitudeamplitude of of the the peak peakI NaINa;; however,however, the the overall overallI-V I-Vrelationship relationship with withthe current the current was notwas altered. not altered. Furthermore, Furthermore, the activationthe activation and and inactivation inactivation time time courses courses of the of peakthe peak INa were clearly modified in the presence of BRV. (C) Concentration-dependent inhibition of INa were clearly modified in the presence of BRV. (C) Concentration-dependent inhibition of peak peak INa caused by different BRV concentrations (mean ± SEM; n = 7). Peak INa was activated by INa caused by different BRV concentrations (mean ± SEM; n = 7). Peak INa was activated by rapid rapid depolarization from −80 to −10 mV. The vertical broken line was placed on the IC50 value of depolarization from −80 to −10 mV. The vertical broken line was placed on the IC50 value of BRV BRV used to show the inhibition of peak INa amplitude in response to the depolarizing command used to show the inhibition of peak I amplitude in response to the depolarizing command voltage. voltage. Na

3.5. Steady-State Inactivation Curve of Peak INa Taken with or without Addition of BRV 3.5. Steady-State Inactivation Curve of Peak INa Taken with or without Addition of BRV The following experiments were undertaken to investigate the effects of BRV on the The following experiments were undertaken to investigate the effects of BRV on the steady-state inactivation of INa. In this set of whole-cell recordings, a two-pulse protocol steady-state inactivation of INa. In this set of whole-cell recordings, a two-pulse protocol was used (Figure5A), and the inactivation parameters of peak INa were then estimated Na wasin the used absence (Figure or 5A), presence and the of in 10activationµM BRV. parameters The normalized of peak amplitude I were then of peak estimatedINa (i.e., in I/Imax) versus the conditioning potential was then derived, as presented in Figure5B . Thereafter, the experimental data were least-squares ﬁtted (indicated in the continuous smooth line) to a Boltzmann function elaborated in the Materials and Methods Section (Figure5B ). Based on the experimental observations, in the control (i.e., BRV was not present), V1/2 = −35.5 ± 2.9 mV, q = 3.8 ± 0.2 e (n = 7), whereas in the presence of 10 µM BRV, V1/2 = −44.3 ± 3.1 mV, q = 3.7 ± 0.2 e (n = 7). Noticeably, as cells were exposed to 10 µM BRV, the midpoint of the steady-state inactivation curve was shifted in a hyperpolarizing direction by approximately 9 mV, regardless of its inability to alter the estimated gating charge (i.e., q value) of the curve. The results from these experiments made it possible to indicate that exposure to BRV is capable of altering the inactivation curve of INa. Biomedicines 2021, 9, x FOR PEER REVIEW 10 of 19

the absence or presence of 10 µM BRV. The normalized amplitude of peak INa (i.e., I/Imax) versus the conditioning potential was then derived, as presented in Figure 5B. Thereafter, the experimental data were least-squares fitted (indicated in the continuous smooth line) to a Boltzmann function elaborated in the Materials and Methods Section (Figure 5B). Based on the experimental observations, in the control (i.e., BRV was not present), V1/2 = −35.5 ± 2.9 mV, q = 3.8 ± 0.2 e (n = 7), whereas in the presence of 10 µM BRV, V1/2 = -44.3 ± 3.1 mV, q = 3.7 ± 0.2 e (n = 7). Noticeably, as cells were exposed to 10 µM BRV, the midpoint of the steady-state inactivation curve was shifted in a hyperpolarizing direction by approximately 9 mV, regardless of its inability to alter the estimated gating charge (i.e., q Biomedicines 2021, 9, 369 10 of 19 value) of the curve. The results from these experiments made it possible to indicate that exposure to BRV is capable of altering the inactivation curve of INa.

Figure 5. BRV-induced modifications to the steady-state inactivation curve of peak INa recorded 2+ from GH3 cells. In this set of experiments, the cells were immersed in a Ca -free Tyrode’s solution Figurecontaining 5. BRV-induced 10 mM tetraethylammoniummodifications to the steady chloride,-state where inactivation the pipette curve was offilled peak withINa recorded Cs+-containing from GH3 cells. In this set of experiments, the cells were immersed in a Ca2+-free Tyrode’s solution solution. (A) Representative I traces obtained without (upper view) and with addition of 10 µM containing 10 mM tetraethylammoniumNa chloride, where the pipette was filled with Cs+-containing BRV (lower view). The uppermost part indicates the voltage profile used, where the different colors solution. (A) Representative INa traces obtained without (upper view) and with addition of 10 µM BRVcorrespond (lower view). to The the differentuppermost command part indicates voltages; the voltage (B) Steady-state profile used, inactivation where the curve different of peak col-INa in µ ± ors correspondthe absence to ( the) and different presence command ( ) of voltages; 10 M BRV (B) (meanSteady-stateSEM; inactivationn = 7). It shouldcurve of be peak noted INa that a in thesignificant absence ( leftward■) and presence (i.e., hyperpolarizing) (○) of# 10 µM BRV shift (mean along ± the SEM; voltage n = 7). axis It should in the inactivation be noted that curve a of the significantcurrent leftward was detected (i.e., hyperpolarizing) in the presence ofshift 10 alµMong BRV), the voltage while the axis slope in the (i.e., inactivation gating charge curve [ qof]) of the the currentcurve remained was detected unaffected. in the presence of 10 µM BRV), while the slope (i.e., gating charge [q]) of the curve remained unaffected. 3.6. Stimulatory Effect of BRV on the Activity of Large-Conductance Ca2+-Activated K+ (BK ) Channels 3.6. StimulatoryCa Effect of BRV on the Activity of Large-Conductance Ca2+-Activated K+ (BKCa) Channels Next, an effort was made to explore whether the presence of BRV could produce any perturbationsNext, an effort on was the probabilitymade to explore of which whether BKCa thechannels presence would of BRV be actively could produce open. Cells any were bathed in high-K+ solution (i.e., 130 mM K+) that contained 1 µM Ca2+, and the recording perturbations on the probability of which BKCa channels would be actively open. Cells pipette was filled with the K+-containing solution. When the inside-out configuration was were bathed in high-K+ solution (i.e., 130 mM K+) that contained 1 µM Ca2+, and the re- firmly established, the excised membrane was voltage-clamped at +60 mV. As depicted in cording pipette was filled with the K+-containing solution. When the inside-out configu- Figure6A, the activity of the BK channels was drastically increased as the intracellular ration was firmly established, the excisedCa membrane was voltage-clamped at +60 mV. As leaflet of the detached patch was exposed to 10 µM of BRV. For example, the presence depicted in Figure 6A, the activity of the BKCa channels was drastically increased as the of resulted in a clear increase in the channel open-state probability from 0.083 ± 0.007 to intracellular leaflet of the detached patch was exposed to 10 µM of BRV. For example, the 0.138 ± 0.011 (n = 8, p < 0.05); however, it was observed that 10 µM of BRV was unable to presence of resulted in a clear increase in the channel open-state probability from 0.083 ± alter the single-channel amplitude. When the BRV was washed out, the channel activity returned to 0.094 ± 0.008 (n = 6, p < 0.05). Alternatively, in the continued presence of BRV, the subsequent addition of GAL-021 or PF1022A reversed the BRV-mediated decrease in BKCa-channel activity; however, further application of TRAM39 failed to exert any effects (Figure6B). TRAM39 was previously reported to be an inhibitor of intermediate- conductance Ca2+-activated K+ channels [66], whereas GAL-021 or PF1022A alone was shown to effectively suppress BKCa-channel activity [64,67]. Biomedicines 2021, 9, x FOR PEER REVIEW 11 of 19

Biomedicines 2021, 9, x FOR PEER REVIEW 11 of 19

0.007 to 0.138 ± 0.011 (n = 8, p < 0.05); however, it was observed that 10 µM of BRV was 0.007 to 0.138 ±unable 0.011 to(n alter= 8, pthe < 0.05);single-channel however, amplitude.it was observed When that the BRV10 µM was of washedBRV was out, the channel unable to alter activitythe single-channel returned to amplitude. 0.094 ± 0.008 When (n = the6, p BRV< 0.05). was Alternatively, washed out, thein the channel continued presence activity returnedof BRV,to 0.094 the ± subsequent 0.008 (n = 6, addition p < 0.05). of Alternatively, GAL-021 or PF1022A in the continued reversed presence the BRV-mediated de- of BRV, the subsequentcrease in additionBKCa-channel of GAL- activity;021 or however, PF1022A furtherreversed application the BRV-mediated of TRAM39 de- failed to exert crease in BKCa-channel activity; however, further application of TRAM39 failed to exert Biomedicines 2021, 9, 369 any effects (Figure 6B). TRAM39 was previously reported to be an inhibitor of intermedi-11 of 19 any effects (Figureate-conductance 6B). TRAM39 Ca was2+-activated previously K+ channelsreported [66],to be whereas an inhibitor GAL-021 of intermedi- or PF1022A alone was 2+ + ate-conductanceshown Ca -activated to effectively K channels suppress [66], BKCa whereas-channel GAL-021 activity [64,67].or PF1022A alone was shown to effectively suppress BKCa-channel activity [64,67].

2+ 2+ + + FigureFigure 6. Stimulatory6. Stimulatory effect effect of BRVof BRV on theon the activity activity of theof the large-conductance large-conductance Ca Ca-activated-activated K K(BK (BKCa)Ca channels) channels in in GH GH3 cells.3 cells. Figure 6. StimulatoryIn thisIn this set effect set of inside-outof of inside-out BRV on current the current activity recordings, recordings, of the thelarge-conductance the cells cells were were immersed immersed Ca2+-activated in a in high-K a high-K K+ (BKsolution+ solutionCa) channels containing containing in GH 1 µ3 M1cells. µM Ca 2+Ca, where2+, where the the + 2+ In this set of inside-outholding current potential recordings, was clamped the cells at +60 were mV. immersed (A) Representative in a high-K BK solutionCa-channel containing traces (blue 1 µM color) Ca obtained, where the in the control (i.e., holding potential was clamped at +60 mV. (A) Representative BKCa-channel traces (blue color) obtained in the control (i.e., holding potentialBRV was was clamped not present) at +60 mV. (left ( Aside) Representative of the figure) BK andCa-channel after the traces bath (blueaddition color) of obtained10 µM of in BRV the control(right side (i.e., of the figure). It BRV was not present) (left side of the figure) and after the bath addition of 10 µM of BRV (right side of the figure). It should BRV was not present)should (leftbe noted side ofthat the the figure) upward and deflectiafter theon bathshows addition the channel of 10 µMopening of BRV event. (right The side lower of thepart figure). (red color) It of the figure be noted that the upward deflection shows the channel opening event. The lower part (red color) of the figure shows the should be notedshows that the the upward expanded deflecti recordson takenshows from the thechannel uppermost opening part event. of the The figure; lower (B )part Summary (red color) bar graph of the showing figure the open-state expanded records taken from the uppermost part of the figure; (B) Summary bar graph showing the open-state likelihood shows the expandedlikelihood records of takenthe presence from the of uppermost BKCa channels part inof thethe presencefigure; (B of) Summary BRV (10 µM),bar graph BRV (10showing µM) plus the open-stateTRAM39 (3 µM), BRV (10 µ µ µ µ likelihood of ofthe theµM) presence presence plus GAL-021of BK of BKCa channelsCa (3channels µM), in and the in BRV thepresence presence (10 µM) of BRV ofplus BRV (10 PF1022A (10µM),M), BRV (3 BRV µM)(10 (10µM) (mean M)plus plus± TRAM39SEM; TRAM39 n = (3 7–8). µM), (3 TheM), BRV channel BRV (10 (10 activityM) plus was µM) plus GAL-021GAL-021measured (3 (3µM),µ whenM), and and theBRV BRV membrane (10 (10 µM)µM) pluspatch plus PF1022A was maintained (3(3 µµM)M) (mean at(mean +60± mV.±SEM; SEM; * Significantlyn n= = 7–8). 7–8). The The diffe channel channelrent from activity activity the was control was measured (p < 0.05) when and Ɨ measured whenthesignificantly the membrane membrane patchdifferent patch was wasfrom maintained maintained the 10 µM at BRV-aloneat +60 +60 mV. mV. * Significantlygroup* Significantly (p < 0.05). different diffe rent from from the the control control ( p(p< < 0.05)0.05) andand Ɨ significantly significantly differentdifferent from from the 10 µMµM BRV-alone group ((pp << 0.05). 3.7. Inhibitory Effect of BRV on INa in Hippocampal Neurons 3.7. Inhibitory Effect of BRV on INa in Hippocampal Neurons 3.7. InhibitoryBRV has Effect been of reported BRV on INa to inproduce Hippocampal significant Neurons changes in the functional activities of BRV has beenneuronsBRV reported has or neural been to produce reported networks significant to that produce include changes significant the hippocampus in the changes functional in [12,29,39,43,47]. the activities functional of To activities investigate of neurons or neuralneuronsthis networksresult, or neural experiments that networks include were the that undertakenhippocampus include the to [12,29,39,43,47].determine hippocampus whether [12 To,29 investigatethe,39 I,43Na ,residing47]. To investi-in mHip- this result, experimentsgatepoE-14 this result,cells were [68] experiments undertaken could be subj wereto ectdetermine undertakento any modifications whether to determine the I byNa residing BR whetherV. Whole-cell in mHip- the INa currentresiding record- in poE-14 cells mHippoE-14[68]ings could were be conducted subj cellsect [68 to] couldanyin cells modifications be that subject were to bathed by any BR modifications V.in Whole-cellCa2+-free Tyrode’s bycurrent BRV. solution,record- Whole-cell and current the elec- ings were conductedrecordingstrode was inwere cells filled conductedthat with were a Cs bathed in+-containing cells in that Ca were2+ solution.-free bathed Tyrode’s As in the Ca solution, cells2+-free were Tyrode’s and exposed the elec- solution, to different and the con- + + trode was filledelectrodecentrations with a was Cs -containingof filled BRV, with thea solution.amplitude Cs -containing As of the the cells solution. peak were INa Aswasexposed the activated cells to different were in response exposed con- totodifferent a brief de- centrations ofconcentrations BRV,polarizing the amplitude pulse of BRV, (Figure of the the 7A,B). amplitude peak For INa wasexample, of the activated peak BRVINa inatwas responsea concentration activated to a inbrief responseof 3de- µM lessened to a brief the polarizing pulsedepolarizingamplitude (Figure 7A,B). of pulse peak For (Figure INa example, from7A,B). 1.97 BRV For± 0.14 example,at toa concentration0.95 ± BRV 0.08 atnA a of( concentrationn 3= µM8, p

0.007 to 0.138 ± 0.011 (n = 8, p < 0.05); however, it was observed that 10 µM of BRV was unable to alter the single-channelBiomedicines amplitude. 2021, 9, x FOR When PEER the REVIEW BRV was washed out, the channel 12 of 19 activity returned to 0.094 ± 0.008 (n = 6, p < 0.05). Alternatively, in the continued presence of BRV, the subsequent addition of GAL-021 or PF1022A reversed the BRV-mediated de- BiomedicinesBiomedicines 20212021, 9, ,x9 ,FOR 369 PEER REVIEW 12 of12 19 of 19 crease in BKCa-channel activity; however, further application of TRAM39 failed to exert any effects (Figure 6B). TRAM39 was previously reported to be an inhibitor of intermediate-conductance Ca2+-activated K+ channels [66], whereas GAL-021 or PF1022A alone was shown to effectively suppress BKCa-channel activity [64,67].

Figure 6. Stimulatory effect of BRV on the activity of the large-conductance Ca2+-activated K+ (BKCa) channels in GH3 cells. In this set of inside-out current recordings, the cells were immersed in a high-K+ solution containing 1 µM Ca2+, where the holding potential was clamped at +60 mV. (A) Representative BKCa-channel traces (blue color) obtained in the control (i.e., Figure 7. Inhibitory effect of BRV on INa in mHippoE-14 hippocampal neurons. (A) Representative BRV was not present) (left side of the figure) and after the bath addition of 10 µM of BRV (right side of the figure). It FigureINa 7.tracesInhibitory obtained effect in the of BRV absence on I (a, inblue mHippoE-14 color) and hippocampalpresence (b, red neurons. color) of (A 3) RepresentativeµM BRV. The up- should be noted that the upward deflection shows the channel opening event. The lower part (red color) of the figure Na Figure 7.per Inhibitory part is the effect voltage of BRV protocol on INa delivered; in mHippoE-14 (B) Summary hippocampal bar graph neurons. showing (A)µ theRepresentative effects of BRV and shows the expanded records taken from the uppermost part of the figure; (B) SummaryINa bartraces graph obtained showing in the the absence open-state (a, blue color) and presence (b, red color) of 3 M BRV. The upper INa tracesBRV obtained plus tefluthrin in the absence (10 µM) (a, on blue the color) peak andamplitude presence of (b,INa redin these color) cells of 3(mean µM BRV. ± SEM; The n up- = 8). * Sig- likelihood of the presence of BKCa channels in the presence of BRV (10 µM), BRV (10part µM) is plus the voltageTRAM39 protocol (3 µM), delivered; BRV (10 (B) Summary bar graph showing the effects of BRV and BRV per partnificantly is the voltage different protocol from delivered; control (p (

Current amplitude (pA) 16 18 20 22 24 Current amplitude (pA) 16 18 20 22 24 the activation and inactivation time courses80 of the current were80 not modified in its pres- 40 40 40 40 ence. 0 0 0 0

Current amplitude (pA) 16 18 20 22 24 Current amplitude (pA) 16 18 20 22 24 80 24 26 2880 30 24 26 28 30 40 sec 40 sec 0 0 24 26 28 30 24 26 28 30 Figure 8. Stimulatory effects of BRV on the activity of BKCa channels identified in mouse mHippoE-14 hippocampal neurons. Figure 8. Stimulatorysec effects of BRVsec on the activity of BKCa channels identified in mouse mHippoE-14 hippocampal neu- In these experiments, cells were bathed in high K+ solution containing 1 µM of Ca2+; inside-out current recordings were rons. In these experiments, cells were bathed in high K+ solution containing 1 µM of Ca2+; inside-out current recordings performed, and channel activity was measured at a holding potential of +60 mV. (A) Representative channel current traces Figure 8.were Stimulatory performed, effects and channelof BRV on activity the activity was me ofasured BKCa channels at a holding identified potential in moof +60use mV.mHippoE-14 (A) Representative hippocampal channel neu- current rons.obtained Intraces these in obtainedexperiments, the control in periodthe cells control (left,were period blue bathed color) (left, in andhighblue after Kcolor)+ solution bath and addition after containing bath of10 addition mM1 µM BRV of of Ca 10 (red2+ mM; color).inside-out BRV Channel-opening (red current color). recordings Channel-opening event is weredenoted performed,event as is an denoted upwardand channel as deflection an activityupward (i.e., was defl outward meectionasured current); (i.e., at outward a holding (B) Summary current); potential bar (B graphof) Summary +60 showingmV. ( Abar) effectsRepresentative graph of showing BRV on channel theeffects probability currentof BRV ofon the traceschannel obtained opening in the during control exposure period to (left, 10 or blue 30 mM color) of BRVand after (mean bath± SEM; additionn =8). of *10 Significantly mM BRV (red different color). from Channel-opening control (p < 0.05). event is denoted as an upward deflection (i.e., outward current); (B) Summary bar graph showing effects of BRV on the Biomedicines 2021, 9, x FOR PEER REVIEW 13 of 19

probability of channelBiomedicines opening2021, during9, 369 exposure to 10 or 30 mM of BRV (mean ± SEM; n = 8). * Significantly different from 13 of 19 control (p < 0.05).

3.9. Simulated Firing of Action Potentials (APs) in Modeled Neurons Used to Mimic the Effect of BRV 3.9. Simulated Firing of Action Potentials (APs) in Modeled Neurons Used to Mimic the Effect of BRV For further evaluation of the effect of BRV on neuronal excitability, we explored how the firing of APs in a modeledFor further neuron evaluation can be adjusted of the effect by ofadding BRV onBRV. neuronal The descriptions excitability, we explored how of this modeled neuronthe firing were of detailed APs in previo a modeledusly neuron[53], and can the be formula adjusted for by a addinghyperpolari- BRV. The descriptions of zation-activated cationthis modeledcurrent was neuron incorporat were detaileded into the previously model [54]. [53], The and parameters the formula used for a hyperpolarization- in this work are illustratedactivated incation Table current1. In attempts was incorporated to study AP intofiring, the a modellong depolarizing [54]. The parameters used in current with 1, 1.5, thisor 2 mA/cm work are2 was illustrated applied into Tablethis modeled1. In attempts neuron. to It study is clear AP from firing, these a long depolarizing 2 simulations that, withcurrent different with 1,current 1.5, or stimulus 2 mA/cm strengths,was applied the presence to this modeled of 10 µM neuron. of BRV It is clear from these µ (i.e., with arbitrarysimulations changes in the that, different with different types of current ionic stimuluscurrents demonstrated strengths, the presenceabove) of 10 M of BRV led to decreases in (i.e.,the AP with firing arbitrary frequency changes as inwell the as different the AP types amplitude of ionic of currents the modeled demonstrated above) led to decreases in the AP firing frequency as well as the AP amplitude of the modeled neuron neuron (Figure 9). (Figure9).

Figure 9. Simulated firing of action potentials (APs) generated from the modeled neuron to mimic Figure 9. Simulated firingthe effects of action of BRV. potentials AP traces (APs) in generated the upper from and lowerthe modeled part of neuron the figure to mimic indicate those theoretically the effects of BRV. APsimulated traces in the to mimicupper and the controllower part condition of the fi ingure which indicate BRV those was theoretically not present and the experimental simulated to mimic thecondition control incondition the presence in which of BRV BRV (10 wasµM), not respectively. present and The the APexperimental firing in each con- panel was evoked by a dition in the presence500 of BRV ms current (10 µM), stimulus respective at aly. strength The AP offiring 1, 1.5 inand each 2 panelµA/cm was2. Theevoked values by a used for this numerical 2 500 ms current stimulussimulation at a strength are illustrated of 1, 1.5 and in Table 2 µA/cm1. . The values used for this numerical simulation are illustrated in Table 1. 3.10. Effects of BRV versus LEV on Acute Seizures in Different Animal Models 3.10. Effects of BRV versusIn LEV a final on Acute series Seizures of studies, in Different we evaluated Animal Models the effects of BRV versus LEV on acute In a final seriesseizures of studies, in different we evaluated animal the models, effects of including BRV versus a sodium LEV on channel acute sei- agonism-based OD-1 zures in different animalmodel models, [55] and including the well-established a sodium channel lithium-pilocarpine-induced agonism-based OD-1 model epilepsy model. We have [55] and the well-establishedrecent characterized lithium-pilocarpin OD-1, ae-induced scorpion toxin,epilepsy which model. could We produce have recent a concentration-, time-, characterized OD-1,and a scorpion state-dependent toxin, which rise in could the peak produce amplitude a concentration-, of INa, shifting time-, the andINa inactivation curve state-dependent riseto in a lessthe negativepeak amplitude potential of and INa, increasingshifting the the INa frequency inactivation of spontaneous curve to a action currents. It less negative potentialcould and generate increasing a significantly the frequency higher of frequency spontaneous of spontaneous action currents. seizures It and epileptiform could generate a significantlydischarges higher compared frequenc with lithium-pilocarpine-y of spontaneous seizures or kainic and acid-induced epileptiform epilepsy, with com- discharges comparedparable with pathological lithium-pilocarpine- changes [55or]. kainic We found acid-induced in the OD-1 epilepsy, model thatwith BRV had significant comparable pathologicaleffects changes on the sustained [55]. We timefound of in severe the OD-1 seizures model (stage that 4 BRV and above,had signif- seconds) (NS: 450 ± 25, icant effects on the sustainedLEV: 390 ± time22, of BRV: severe 220 seizures± 20) (p (stage< 0.05) 4 compared and above, to seconds) the control (NS: group 450 (Figure 10A). The BRV group also had a significantly lower number of animals with severe seizures (NS: 60%, LEV: 45%, BRV: 35%) (p < 0.05) and severe seizure counts within the 8 h study duration (NS: 35, LEV: 24, BRV: 19) (p < 0.05) as compared to the control group (Figure 10B,C). In comparison, in the lithium-pilocarpine-induced epilepsy model, compared to the control Biomedicines 2021, 9, x FOR PEER REVIEW 14 of 19

± 25, LEV: 390 ± 22, BRV: 220 ± 20) (p < 0.05) compared to the control group (Figure 10A). The BRV group also had a significantly lower number of animals with severe seizures (NS: 60%, LEV: 45%, BRV: 35%) (p < 0.05) and severe seizure counts within the 8 h study Biomedicines 2021, 9,duration 369 (NS: 35, LEV: 24, BRV: 19) (p < 0.05) as compared to the control group (Figure 14 of 19 10B,C). In comparison, in the lithium-pilocarpine-induced epilepsy model, compared to the control group, both LEV and BRV had significant effects on the latency of stage 3 seizures and above, NS: 25.8 ± 1.5, LEV: 35 ± 1.8, BRV: 37 ± 2) (p < 0.05) (Figure 10D), the group, both LEV and BRV had signiﬁcant effects on the latency of stage 3 seizures and number of animals with severe seizures (NS: 58%, LEV: 38%, BRV: 36%) (p < 0.05) (Figure above, NS: 25.8 ± 1.5, LEV: 35 ± 1.8, BRV: 37 ± 2) (p < 0.05) (Figure 10D), the number of 10E), and mortalityanimals (NS: with 27%, severe LEV: seizures 17%, BRV: (NS: 15%) 58%, (p LEV: < 0.05) 38%, (Figure BRV: 36%)10F). (Thep < 0.05)observa- (Figure 10E), and tions in these mortalityanimal models (NS: 27%, demonstrated LEV: 17%,BRV: the differential 15%) (p < 0.05) effects (Figure of BRV 10F). and The LEV observations on in these acute seizuresanimal and neuronal models hyperexcitability. demonstrated the differential effects of BRV and LEV on acute seizures and neuronal hyperexcitability.

Figure 10. EffectsFigure of 10. BRV Effects versus of LEVBRV onversus acute LEV seizures on acute in OD-1 seizures and lithium-pilocarpinein OD-1 and lithium-pilocarpine animal models. animal (A–C ) In the OD-1 model, BRVmodels. had a significant (A–C) In effectthe OD-1 on themodel, sustained BRV had time a ofsignificant severe seizures effect on (stage the sustained 4 and above) timecompared of severe to the control group. The BRVseizures group (stage also had4 and a lowabove) number compared of rats to with the severecontrol seizures group. The and BRV a lower group severe also seizure had a count,low num- as compared to the control groupber of (*ratsp < with 0.05, severen = 7 seizures in each group);and a lower (D–F severe) In the seizure lithium-pilocarpine-induced count, as compared to the epilepsy control model, group compared to the control(* group,p < 0.05, both n = LEV7 in each and BRVgroup); had (D significant–F) In the effectslithium-pilocarpine- on the latencyinduced of stage epilepsy 3 seizures, model, the number com- of rats with severe seizures,pared and to mortalitythe control (* group,p < 0.05, bothn =LEV 7 in and each BRV group). had significant The data wereeffects analyzed on the latency using anof stage ANOVA 3 sei- followed by Fisher’s leastzures, significant the number difference of rats tests. with severe seizures, and mortality (* p < 0.05, n = 7 in each group). The data were analyzed using an ANOVA followed by Fisher’s least significant difference tests. 4. Discussion 4. Discussion The principal findings presented in this study are as follows: First, cell exposure to The principal findings presented in this study are as follows: First, cell exposure to brivaracetam (BRV), a bioavailable LEV derivative, resulted in a concentration-dependent brivaracetam (BRV), a bioavailable LEV derivative, resulted in a concentration-dependent inhibition of IK(M); however, it mildly depressed the amplitude of IK(DR) and Ih. Second, inhibition of IK(M); however, it mildly depressed the amplitude of IK(DR) and Ih. Second, the the presence of this agent inhibited the peak amplitude of INa in a concentration-dependent presence of this agent inhibited the peak amplitude of INa in a concentration-dependent manner together with a leftward shift in the steady-state inactivation curve of the current. manner together with a leftward shift in the steady-state inactivation curve of the current. Third, in the case of the inside-out current recordings, addition of BRV to the intracellular Third, in the case of the inside-out current recordings, addition of BRV to the intracellular side of the excised patch enhanced the probability BKCa channels that would be open. side of the excisedFourth, patch in the enhanced mHippoE-14 the probability hippocampal BK neurons,Ca channels BRV that was would effective be open. in suppressing the Fourth, in the mHippoE-14 hippocampal neurons, BRV was effective in suppressing the amplitude of peak INa with minimal changes in the activation and inactivation time courses amplitude of ofpeak the I current.Na with minimal These observations changes in thusthe activation suggest evidence and inactivation that, besides time being a high- courses of theaffinity current. ligand These for observations SV2A [9,15,39 th,41us– 45suggest], BRV canevidence perturb that, the ionicbesides currents being specifieda herein, high-affinity ligandhence for disclosing SV2A [9,15,39,41–45], a potential additional BRV can perturb impact the on ionic the functional currents specified activities of different herein, hence excitabledisclosing cells. a potential additional impact on the functional activities of different excitable cells. The effective IC50 values needed for BRV-induced inhibition of the IK(M) or peak INa The effective IC50 values needed for BRV-induced inhibition of the IK(M) or peak INa observed in the GH3 cells were estimated to be 6.5 or 12.2 µM, respectively. In addition 3 observed in theto GH a measurable cells were reduction estimated in to the be 6.5IK(M) or amplitude12.2 µM, respectively. in response In to addition long-lasting to maintained depolarization, the activation time course of the current became slower in the presence of BRV. The BRV molecule can therefore reach the binding site once the IK(M) channels are overly activated and reside in either the open state or in the open conformation. Addition- allly, despite the decrease in peak INa combined with a lack of the overall IV relationship to the current, the steady-state inactivation curve of the current was found to shift along the voltage axis toward a hyperpolarized potential (about 9 mV), with no measurable per- Biomedicines 2021, 9, 369 15 of 19

turbations on the estimated gating charge related to the conditioning potential versus the relative current amplitude. Consequently, the window INa [70] was expected to decrease in the presence of BRV. By extension, in the inside-out current recordings, the addition of BRV to the internal leaflet of the excised patch conceivably elevated the probability of BKCa- channel openings, notwithstanding its inability to modify the single-channel amplitude. In the continued presence of BRV, subsequent application of either GAL-021 or PF1022A were detected to effectively attenuate increases in channel activity. In previous recent pharmacokinetic studies on BRV, following intravenous adminis- tration of this agent, its plasma concentration was reported to range between 1 and 3 mg/L (i.e., 4.7 and 14.1 µM) [34,71,72]. Therefore, the ionic channels (i.e., M-type (KCNQx)K+, NaV (SCNx), and BKCa (KCNMA1) channels) are a relevant target for the pharmacolog- ical actions of this drug and may virtually occur within the clinically therapeutic range although the detailed mechanism by which BRV interferes with actions on these types of ion channels still requires further detailed investigation. It should be mentioned that glioma cells can functionally express the magnitude of INa, which may be linked to the malignant transformation of neoplastic cells [73]. BRV was also previously reported to exert anti-neoplastic actions identified in glioma cells [40]. In keeping with previous observations [47], it was possible to determine that the presence of BRV can lead to a possible reduction in the amplitude of peak INa identified in GH3 or mHippoE-14 cells although the activation and inactivation time course of the current in response to the depolarizing command voltage remained unperturbed when the cells were exposed to BRV. As such, to what extent BRV-mediated changes in the magnitude of INa residing in glioma cells will participate in its anti-neoplastic actions remains to be established. Alternatively, BRV-induced relief of pain sensation as reported previously [1,2,39] could be partly explained by its inhibition of peak INa in sensory neurons. It is worth noting that Levetiracetam was previously shown to decrease the amplitude of IK(DR) when accompanied by an enhanced inactivation current time course [62]. However, in this study, the amplitude of IK(DR) or Ih was mildly inhibited by adding BRV, and minimal changes in IK(DR) inactivation in response to different levels of sustained depolarization were observed in its presence. It is conceivable, therefore, that in contrast to LEV, the IK(DR) may not be an obligate target with which the BRV molecule can interfere. Moreover, the effects of BRV and LEV on the various types of ionic currents demonstrated herein could not be solely explained by their binding to the synaptic vesicle protein 2A (SV2A) in hippocampal neurons and pituitary cells [41–43,45,74], which was thought to be the case in both synaptic or endocrine vesicle exocytosis and neurotransmitter release, although they have been observed to be high-affinity SV2A ligands [9,15,39,43,44]. In the theoretical study, it was possible to mimic the BRV action on central neurons. It was noted that when cells were exposed to BRV, the simulated frequency of neuronal AP firing elicited in response to different depolarizing stimuli was obviously decreased owing to the changes in the conductance values of the different types of ionic currents referenced above, in combination with the reduced AP amplitude. It is conceivable, therefore, that its effects on neuronal APs occurring in vivo will be affected. There were fundamental differences between the two animal models. In OD-1 model, we needed to anesthetize these animals first to stereotactically inject the OD-1 toxin and observed the seizure parameter 24 h later. There was thus no acute mortality in this group because of the initial anesthetic effect, which reduced the initial acute neuronal excitotoxicity. We evaluated the parameter severe seizures as an indicator of high neuronal excitotoxicity. For pilocarpine model, the acute excitotoxicity was prominent after initial intraperitoneal injection, thus it is not uncommon to lead to acute mortality following stage 4–5 seizure and status epilepticus. The latency to acute stage 3 seizures, number of rats with severe seizures and acute mortality were used to evaluate the high neuronal excitotoxicity. Therefore, the seizure parameters evaluated were different in both models. Biomedicines 2021, 9, 369 16 of 19

The present evaluation of the effects of BRV versus LEV on acute seizure animal models further demonstrated the different ionic effects of BRV and LEV. Compared to LEV, sodium channel modulation of BRV, as demonstrated in the present study, explained its significant effect on OD-1, a unique sodium channel-agonism-based animal model of epilepsy and seizure. However, our previous study on LEV’s ionic mechanism did not reveal the underlying LEV mechanism for sodium channel modulation [62]. Nevertheless, both LEV and BRV had significant effects on the lithium-pilocarpine-induced seizure model, which was in line with previous observations regarding LEV’s effect on this model and the common SV2A mechanism of both medications in this model [75,76]. Furthermore, the various actions associated with the mechanism accounted for the finding that the response in the presence of BRV has been found to be several minutes faster than that with LEV in patients with photosensitive epilepsy [77], and BRV has been shown to be a useful treatment option in patients with epilepsy who have previously failed to respond to or tolerate LEV [48,78]. The unique ionic mechanism, in addition to the common SV2A modulation, justifies the role of BRV in rationale polytherapy for epileptic disorders, in terms of both efficacy and adverse events.

5. Conclusions BRV’s multiple ionic mechanism in electrically excitable cells and a concerted effect on neuronal excitability underlies its therapeutic potential in clinical neuronal hyperexcitability disorders.

Author Contributions: Conceptualization, T.-Y.H., S.-N.W. and C.-W.H.; formal analysis, T.-Y.H., S.-N.W. and C.-W.H.; funding acquisition, S.-N.W. and C.-W.H.; investigation, T.-Y.H., S.-N.W. and C.-W.H.; methodology, S.-N.W. and C.-W.H.; writing—original draft, T.-Y.H., S.-N.W. and C.-W.H.; writing—review and editing, T.-Y.H., S.-N.W. and C.-W.H. All authors have read and agreed to the published version of the manuscript. Funding: This study was partly funded by a grant from Ministry of Science and Technology, Taiwan (Grant number: MOST-105-2320-B-037-013-MY3, 107-2314-B-006-018-, 107-2320-B-006-019-, 108-2320- B-006-023-, 109-2314-B-006 -034 -MY3, and a grant from National Cheng Kung University (Grant number: D106-35A13). Institutional Review Board Statement: All the animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) (Approval No: 110147, Date: 20 February 2021) at National Cheng Kung University, Tainan, Taiwan. Informed Consent Statement: Not applicable. Data Availability Statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments: The authors gratefully acknowledge technical support provided by Kaisen Lee during this work. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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