Effective Perturbations by Phenobarbital on INa, IK(erg), IK(M) and IK(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 (GABAA) 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 (INa) in a concentration-dependent fashion, with an effective IC50 value of 83 µM. The cumulative inhibition of INa, evoked by pulse train stimulation, was enhanced by PHB. However, tefluthrin, an activator of INa, could attenuate PHB-induced reduction in the decaying time constant of INa inhibition evoked by pulse train stimuli. In addition, the erg (ether-à-go-go-related gene)-mediated K+ current (IK(erg)) was also blocked by PHB. The PHB-mediated inhibition on IK(erg) could not be overcome by flumazenil (GABA antagonist) or chlorotoxin (chloride channel blocker). The PHB reduced the recovery of IK(erg) by a two-step voltage protocol with a geometrics-based progression, but it increased the decaying rate of IK(erg), evoked by the envelope-of-tail method. About the M-type K+ currents (IK(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 (IK(DR)) was inhibited by PHB, while the cumulative inhibition of IK(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 GABAA receptors.

Characterization in Potent Modulation on Voltage-Gated Na+ Current Exerted by Deltamethrin, a Pyrethroid Insecticide

“…In this set of measurement, the stimulus protocol, consisting of repetitive depolarization of −10 mV (20 ms in each pulse with a rate of 40 Hz for 1 s), was applied to the tested cells which were voltage-clamped at −80 mV. In accordance with earlier reports [ 22 , 23 , 24 , 25 , 36 ], as demonstrated in Figure 4 and Figure 5 , during the control period (i.e., neither Tef nor DLT was present), the exponential time course of I Na(T) inactivation observed in GH 3 cells was observed during a 1-s repetitive depolarization from −80 to −10 mV, and an evolving decaying time constant of 22.1 ± 2.8 ms ( n = 8) was then yielded. In other words, there appeared to be a progressive current decay (indicated with the dashed arrows in Figure 4 A) with a single-exponential process.…”

“…It has been demonstrated that, prior to being activated during repetitive short pulses, the inactivation of I Na(T) is able to accumulate [ 22 , 23 , 25 , 34 , 35 , 36 ]. For this reason, we next explored if Tef or DLT could modify the extent of I Na(T) activated either during or following the PT depolarizing stimuli.…”

“…The amplitude of I Na(L) obtained during the presence of DLT at a concentration of 100 µM was considered as 100% and we then compared current magnitudes (i.e., I Na(T) and I Na(L) ) during cell exposure to varying concentrations of DLT. The concentration-dependent stimulation by DLT of I Na(T) or I Na(L) observed in GH 3 cells was determined by fitting experimental data set to a modified Hill function [ 10 , 25 ], which can be given as follows. …”

“…Upon rapid depolarization, the Na V channels, by which macroscopic voltage-gated Na + currents ( I Na ) are encoded, are characterized by going through rapid transitions from the closed (resting) state to the open state, and then by swiftly changing to the inactivated state [ 20 , 21 ]. The inactivation of I Na has been also demonstrated to accumulate before being stimulated during repetitive short depolarizing pulses [ 22 , 23 , 24 , 25 ]. Consequently, once being evoked, the increased magnitude of I Na can quickly depolarize the cell membrane through positive feedback cycle and, in turn, elicit the upstroke of the action potentials, thereby intrinsically governing the amplitude, frequency, and/or pattern of firing action potentials, as well as hormonal secretion, in an array of excitable cells [ 20 , 21 , 26 , 27 ].…”