Characterization in Inhibitory Effectiveness of Carbamazepine in Voltage-Gated Na+ and Erg-Mediated K+ Currents in a Mouse Neural Crest-Derived (Neuro-2a) Cell Line

Abstract

Carbamazepine (CBZ, Tegretol®) is an anticonvulsant used in the treatment of epilepsy and neuropathic pain; however, several unwanted effects of this drug have been noticed. Therefore, the regulatory actions of CBZ on ionic currents in electrically excitable cells need to be reappraised, although its efficacy in suppressing voltage-gated Na+ current (INa) has been disclosed. This study was undertaken to explore the modifications produced by CBZ on ionic currents (e.g., INa and erg-mediated K+ current [IK(erg)]) measured from Neuro-2a (N2a) cells. In these cells, we found that this drug differentially suppressed the peak (transient, INa(T)) and sustained (late, INa(L)) components of INa in a concentration-dependent manner with effective IC50 of 56 and 18 μM, respectively. The overall current–voltage relationship of INa(T) with or without the addition of CBZ remained unchanged; however, the strength (i.e., ∆area) in the window component of INa (INa(W)) evoked by the short ascending ramp pulse (Vramp) was overly lessened in the CBZ presence. Tefluthrin (Tef), a synthetic pyrethroid, known to stimulate INa, augmented the strength of the voltage-dependent hysteresis (Hys(V)) of persistent INa (INa(P)) in response to the isosceles-triangular Vramp; moreover, further application of CBZ attenuated Tef-mediated accentuation of INa(P)’s Hys(V). With a two-step voltage protocol, the recovery of INa(T) inactivation seen in Neuro-2a cells became progressively slowed by adding CBZ; however, the cumulative inhibition of INa(T) evoked by pulse train stimulation was enhanced during exposure to this drug. Neuro-2a-cell exposure to CBZ (100 μM), the magnitude of erg-mediated K+ current measured throughout the entire voltage-clamp steps applied was mildly inhibited. The docking results regarding the interaction of CBZ and voltage-gate Na+ (NaV) channel predicted the ability of CBZ to bind to some amino-acid residues in NaV due to the existence of a hydrogen bond or hydrophobic contact. It is conceivable from the current investigations that the INa (INa(T), INa(L), INa(W), and INa(P)) residing in Neuro-2a cells are susceptible to being suppressed by CBZ, and that its block on INa(L) is larger than that on INa(T). Collectively, the magnitude and gating of NaV channels produced by the CBZ presence might have an impact on its anticonvulsant and analgesic effects occurring in vivo.

Characterization in Effective Stimulation on the Magnitude, Gating, Frequency Dependence, and Hysteresis of INa Exerted by Picaridin (or Icaridin), a Known Insect Repellent

“…The tested cell was held at −80 mV, and the stimulus protocol, which was designed to consist of repetitive depolarization to −10 mV (20 ms in each pulse with a rate of 40 Hz for 1 s), was then imposed on it. In accordance with previous observations [ 38 , 40 , 41 , 42 ], as depicted in Figure 4 A–C, during the control period (i.e., picaridin was not present), the process of I Na(T) inactivation detected in GH 3 cells was robustly evoked by a 1 s repetitive depolarization from −80 to −10 mV, and an evolving inactivation time constant of 26.5 ± 2.4 ms (n = 7) was yielded. That is, there was a sudden current decay with a single-exponential process.…”

“…In the present study, the time-dependent decline of I Na(T) activated during a 40 Hz train of depolarizing voltage steps, the protocol of which consisted of 20 ms pulses delivered from −80 to −10 mV at a rate of 40 Hz for a total duration of 1 s, was robustly detected in an exponential fashion, strongly indicating that there is marked use dependence of I Na(T) elicited by repetitive depolarizations, as stated previously [ 39 , 40 , 41 , 42 ]. Moreover, such exponential decrease in I Na(T) activated during pulse train stimulation was noted to become overly blunted with cell exposure to picaridin.…”

“…It has been previously demonstrated that, prior to being activated during repetitieve short pulses, the inactivation of I Na(T) can accumulate [ 27 , 38 , 39 , 40 , 41 ]. Therefore, in subsequent experiments, we proceeded to explore if the presence of picaridin could adjust the inactivation process of I Na(T) during a train of membrane depolarizations.…”

“…Curve-fitting (linear or nonlinear) to the experimental data set applied was fitted by use of manifold analytical tools, that included the Microsoft “Solver” embedded into Excel ® 2022 (Microsoft) and OriginPro ® 2022b program (OriginLab ® ; Microcal; Scientific Formosa, Kaohsiung, Taiwan) [ 40 , 41 ]. The electrophysiological data are presented as the mean ± standard error of the mean (SEM).…”

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

“…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 ].…”