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

Abstract

Picaridin (icaridin), a member of the piperidine chemical family, is a broad-spectrum arthropod repellent. Its actions have been largely thought to be due to its interaction with odorant receptor proteins. However, to our knowledge, to what extent the presence of picaridin can modify the magnitude, gating, and/or the strength of voltage-dependent hysteresis (Hys_(V)) of plasmalemmal ionic currents, such as, voltage-gated Na⁺ current [I_Na], has not been entirely explored. In GH₃ pituitary tumor cells, we demonstrated that with exposure to picaridin the transient (I_Na(T)) and late (I_Na(L)) components of voltage-gated Na⁺ current (I_Na) were differentially stimulated with effective EC₅₀’s of 32.7 and 2.8 μM, respectively. Upon cell exposure to it, the steady-state current versus voltage relationship I_Na(T) was shifted to more hyperpolarized potentials. Moreover, its presence caused a rightward shift in the midpoint for the steady-state inactivate curve of the current. The cumulative inhibition of I_Na(T) induced during repetitive stimuli became retarded during its exposure. The recovery time course from the I_Na block elicited, following the conditioning pulse stimulation, was satisfactorily fitted by two exponential processes. Moreover, the fast and slow time constants of recovery from the I_Na block by the same conditioning protocol were noticeably increased in the presence of picaridin. However, the fraction in fast or slow component of recovery time course was, respectively, increased or decreased with an increase in picaridin concentrations. The Hys_(V)’s strength of persistent I_Na (I_Na(P)), responding to triangular ramp voltage, was also enhanced during cell exposure to picaridin. The magnitude of resurgent I_Na (I_Na(R)) was raised in its presence. Picaritin-induced increases of I_Na(P) or I_Na(R) intrinsically in GH₃ cells could be attenuated by further addition of ranolazine. The predictions of molecular docking also disclosed that there are possible interactions of the picaridin molecule with the hNa_V1.7 channel. Taken literally, the stimulation of I_Na exerted by the exposure to picaridin is expected to exert impacts on the functional activities residing in electrically excitable cells.

1. Introduction

Picaridin (icaridine, 1-piperidinecarboxylic acid 2-(2-hydroxyethyl)-1-methylpropylester), a cyclic amine and a member of the piperidine chemical family, is viewed as a synthetic, broad-spectrum arthropod repellent [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. The repellent and deterrent activities of picaridin have been previously demonstrated to involve olfactory sensing in mosquitoes and ticks, via their interactions with odorant receptor proteins residing in neurons [17,18,19,20,21,22]. However, to the best of our knowledge, the questions of whether and how picaridin, or other relevant compounds, act to cause any modifications on other types of transmembrane ionic currents have not yet been answered.

It has been established that nine isoforms (i.e., Na_V1.1–1.9 [or SCN1A-SCN5A and SCN8A-SCN11A] of voltage-gated Na⁺ (Na_V) channels are distributed in mammalian excitable tissues which are present in the central or peripheral nervous system, as well as in the neuroendocrine system [23,24,25]. Once activated, the increased activity of these channels can quickly depolarize the cell membrane and, in turn, elicit the upstroke of the action potential, thereby intrinsically governing the amplitude, frequency, and/or pattern of firing action potentials in an array of excitable cells [23,24,25,26,27]. Of additional note, some inhibitors of Na_V channels (e.g., KMUP-1 and ranolazine) were demonstrated to increase the inactivation rate of I_Na [28,29,30], while some activations of Na_V channels (e.g., tefluthrin) could preferentially retard the inactivation rate, as well as enhance the transient (I_Na(T)) and late (I_Na(L)) components of I_Na [31,32]. KMUP-1 was viewed as a xanthine and piperazine derivative [30], and ranolazine as a piperazine derivative, which is structurally similar to trimetazidine and known to be a blocker of I_Na(L) [28,29,33,34], while tefluthrin has been reported to be a synthetic type-I pyrethroid used to kill a wide range of insects [35,36]. However, whether and how picaridin can affect the magnitude, gating kinetics, and/or voltage-dependent hysteresis (Hys_(V)) of I_Na remains mostly obscure, although it is an insect repellent which belongs to the piperidine chemical family.

Due to the reasons elaborated above, the overall objective of the current investigation was to explore if picaridin, and other relevant compounds, could exert any adjustments on the magnitude, gating, use dependence, and/or Hys_(V) behavior of plasmalemmal ionic currents, particularly voltage-gated Na⁺ current (I_Na). The biophysical and pharmacological properties of I_Na, including transient I_Na (I_Na(T)), late I_Na (I_Na(L)), persistent I_Na (I_Na(P)) and resurgent I_Na (I_Na(R)), were extensively studied in pituitary GH₃ lactotrophs. Findings from the present studies provide the notion that picaridin is capable of perturbing the magnitude, gating, and/or Hys_(V) strength of I_Na(T) in concentration-, time-, frequency- and Hys_(V)-dependent manners.

2. Results

2.1. Stimulatory Effect of Picaridin on Voltage-Gated Na⁺ Current (I_Na) Experimentally Recorded from Pituitary GH₃ Cells

In the start of experiments, we attempted to explore if cell exposure to picaridin produced any modifications on the magnitude of I_Na identified in these cells. To verify the appearance of I_Na, we used Ca²⁺-free Tyrode’s solution as a bathing medium in which 10 mM tetraethylammonium chloride (TEA) and 0.5 mM CdCl₂ were contained, and the recording electrode was then filled up with a Cs⁺-containing solution. As illustrated in Figure 1A, one minute after cells were exposed to picaridin (3 or 10 μM), the I_Na magnitude (with a rapidly activating and inactivating property) elicited by 30-ms depolarizing step from −100 to −10 mV became progressively increased. For example, one minute after cell exposure to 3 or 10 μM, the peak (or transient, I_Na(T)) component of I_Na was respectively raised to 828 ± 44 pA (n = 8, p < 0.05) or 900 ± 50 pA (n = 8, p < 0.05) from a control value of 712 ± 39 pA (n = 8, p < 0.05). The observed picaridin-mediated increase of I_Na(T) was also concurrently accompanied by a striking retardation in the inactivation time course of the current, as evidenced by a striking increase in the value of the slow component of the inactivation time constant (τ_inact(S)) from 12 ± 2 to 19 ± 4 ms (n = 8, p < 0.05). After washout of picaridin, current magnitude returned to 719 ± 41 pA (n = 8, p < 0.05). Moreover, picaridin-mediated stimulation of I_Na was also counteracted by further addition of tetrodotoxin (TTX, 1 μM) or ranolazine (Ran, 10 μM). TTX or Ran was reported to suppress I_Na in pituitary lactotrophs [26,28,37].

The relationship between the picaridin concentration and the percentage increase of I_Na(T) or I_Na(L) was further constructed (Figure 1B). Addition of picaridin was noticed to increase the I_Na(T) or I_Na(L) amplitude in a concentration-dependent fashion. When the experimental data became least-squares fitted to a Hill function, as detailed in Materials and Methods, the half-maximal concentration (i.e., EC₅₀) required for the stimulatory effect of picaridin on I_Na(T) or I_Na(L) observed in these cells was yielded as 32.7 or 2.8 μM, respectively. Overall, the data from these experiments allowed us to ascertain that the presence of picaridin caused a stimulatory but differential effect on I_Na(T) and I_Na(L) in a concentration-dependent manner.

2.2. Effect of Picaridin on Mean Current versus Voltage (I–V) Relationship of I_Na(T)

We next examined the perturbations of this compound on I_Na(T) measured at various membrane potentials. As depicted in Figure 2, a steady-state I–V relationship of I_Na(T) with or without the picaridin presence was constructed in these cells. The application of 10 μM picaridin resulted in a striking increase in the amplitude of I_Na(T) elicited by depolarizing steps. For example, as the examined cells were rapidly depolarized from −80 to −20 mV, the addition of picaridin (10 μM) noticeably raised the I_Na(T) magnitude from 744 ± 49 to 940 ± 56 pA (n = 7, p < 0.05). Moreover, the steady-state I–V relationship of I_Na(T) was found to be shifted to more negative potentials during GH₃-cell exposure to 10 μM picaridin.

2.3. Characterization of Picaridin-Induced Modifications on the Steady-State Inactivation Curve of I_Na(T)

To further characterize the stimulatory effect of picaridin on I_Na(T) in response to an abrupt depolarization pulse, we continued to examine the steady-state inactivation curve of the current by using a two-step voltage protocol. Figure 3 shows the steady-state inactivation curve of I_Na(T) acquired with or without exposure to picaridin (10 μM). In this series of experiments, a 30 ms conditioning pulse was given to various membrane potentials to precede the test pulse (30 ms in duration) to −10 mV from a holding potential of −80 mV. The interval between two sets of voltage pulses applied was about 1 min to allow complete recovery of I_Na(T). The relationship between the conditioning potentials and the normalized amplitudes of I_Na(T) with or without the picaridin addition were plotted and, then, appropriately fitted to a Boltzmann function, detailed under Materials and Methods. In control, V_1/2 = −48 ± 2 mV, q = 3.5 ± 0.6 e (n = 7), whereas in the presence of 10 μM picaridin, V_1/2 −43 ± 2 mV, q = 3.6 ± 0.6 e (n = 7). It meant, therefore, that the presence of this compound not only elevated the maximal magnitude of I_Na(T), but it also shifted the steady-state inactivation curve of I_Na(T) to more depolarized potential (i.e., in the rightward direction) by approximately 5 mV. However, we found no appreciable adjustment in the q (apparent gating charge) value of the inactivation curve in its presence. These results reflected that the presence of picaridin could increase the I_Na(T) magnitude in a voltage-dependent fashion in GH₃ cells.

2.4. Picaridin-Induced Retardation in Cumulative Inhibition of I_Na(T) Inactivation during Rapid Depolarizing Stimuli

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. 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 4A–C, during the control period (i.e., picaridin was not present), the process of I_Na(T) inactivation detected in GH₃ 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. It is also noteworthy that during cell exposure of picaridin at a concentration of 3 or 10 μM, the time constant of I_Na(T) decay evoked by the same train of depolarizing voltage pulses was, respectively, raised to 48.1 ± 2.6 ms (n = 7, p < 0.05) or 63.5 ± 2.8 ms (n = 7, p < 0.05), in addition to a considerable increase in I_Na(T) amplitude. Moreover, upon continued presence of 10 μM picaridin, a further application of brivaracetam (BRV, 10 μM) noticeably attenuated the picaridin-prolonged time constant of I_Na(T) inactivation during repetitive depolarizations to 41.1 ± 2.6 ms (n = 7, p < 0.05). BRV was recently reported to suppress I_Na(T) amplitude [43]. Overall, these results meant that, apart from the increase in I_Na(T) amplitude, with cell exposure to picaridin the decrease in the decaying of I_Na(T) in response to a 1-s train of depolarizing pulses (i.e., accumulative inactivation of I_Na(T)) could become retarded in these cells.

2.5. Effect of Picaridin on the Recovery Time Course of I_Na(T) Inactivation after the Conditioning Train of Depolarizing Stimuli

The earlier investigations demonstrated a distinguishing type of recovery process from I_Na(T) inactivation, as elicited by a train of conditioning depolarizing stimuli ([38,41,44]. In light of these previous studies, we next sought to investigate if such a recovery process as that emerging in I_Na(T) could be modified by adding picaridin. In this separate set of experiments, the preceding condition train given was composed of 40 20-ms pulses separated by 5 ms intervals at −80 mV for a duration of 1 s. Following such conditioning train, a two-step voltage protocol, where the recovery process was measured, was further imposed on the tested cells to evoke I_Na(T). That is, the I_Na(T) was initially induced by a 30 ms step from −80 to −10 mV, voltage was thereafter returned to −80 mV for a variable length of time in a geometric progression with a common ratio of 2 (Figure 5A). In keeping with previous investigations, the resulting recovery time course, which slowed inactivation of I_Na(T) in response to the conditioning depolarizing stimuli, was noticed to emerge largely in a biphasic manner (i.e., fast and slowly recovering pools), while the recovery time course of current inactivation with no preceding conditioning train pulse was fitted in a satisfactory way by a single exponential. However, when the conditioning train pulse stimulation was applied (Figure 5B), during the control period, the experimental data were optimally fit with a sum of two exponential functions, which was characterized by fast (τ_fast) and slow time constants (τ_slow). Additionally, with cell exposure to picaridin, the evolving values of both τ_fast and τ_slow were noted to rise, while the fraction in the fast component increased and that in the slow component decreased.

Table 1. Summary of results showing the parameter values for effects of picaridin on the recovery of the I_Na block during the preceding train pulse. These parameters are elaborated in detail in Materials and Methods. All values are mean ± SEM. * Significantly different from controls (p < 0.05).

	N	τfast (ms)	τslow (ms)	A	B
Control	8	12.1 ± 0.5	801 ± 12	0.62 ± 0.04	0.43 ± 0.02
Picaridin (3 μM)	8	13.9 ± 0.6 *	893 ± 14 *	0.67 ± 0.04 *	0.36 ± 0.02 *
Picaridin (10 μM)	8	15.1 ± 0.7 *	976 ± 15 *	0.72 ± 0.04 *	0.29 ± 0.02 *

summarizes the values of numerical parameters used for estimation of nonlinear recovery time course in the absence and presence of picaridin (3 or 10 μM). The results allowed us to propose that, in GH₃ cells, the I_Na activated by a conditioning train of pulse could cause a large fraction of Na_V channels to shift toward the slowly recovering pool, as demonstrated in previous studies [38,44]. Moreover, of interest, during exposure to picaridin, the fraction of the slowly recovering pool of Na_V channels during the conditioning train pulse became smaller, while that of the fast recovery pool of the channels tended to be greater; however, both τ_fast and τ_slow values of recovery time course rose in its presence.

2.6. Effect of Picaridin on the Strength of Voltage-Dependent Hysteresis (Hys_(V)) of Persistent I_Na (I_Na(P)) Elicited by an Upright Isosceles-Triangular V_ramp

The Hys_(V) behavior of I_Na(P) has been recently demonstrated with a figure-of-eight (i.e., ∞-shaped) configuration as current trace was induced by an upright isosceles-triangular V_ramp [33,37,45,46]. Any modifications on I_Na(P) were expected to have a high impact on membrane excitability [47,48,49,50,51]. In this regard, we continued to study if the picaridin existence could modify the Hys_(V) strength in response to the upright isosceles-triangular V_ramp. The measurements were undertaken in cells bathed in Ca²⁺-free Tyrode’s solution, and we filled up the electrodes with a solution containing Cs⁺. The tested cells were held at −80 mV and an upright isosceles-triangular V_ramp, ranging between −100 and +50 mV for a duration of 1.6 s (i.e., ramp speed of ±0.25 mV/ms), was imposed on them. As demonstrated in Figure 6A,B in the control period, current traces in response to such triangular V_ramp evolved to exhibit two types of hysteretic loops (i.e., low- and high-threshold loops). The peak amplitude at low- and high-threshold loop appeared at around −80 and −20 mV, respectively, while current trajectory at low- and high-threshold loop occurred in a clockwise and anti-clockwise direction, respectively. As shown in Figure 6B, upon cell exposure to picaridin, the strength of I_Na(P)’s Hys_(V) became overly increased. For example, upon cell exposure to this compound at a concentration of 10 μM, the I_Na(P) amplitudes at either high-threshold loop (i.e., at the level of −20 mV) or low-threshold loop (i.e., at the level of −80 mV), respectively, increased from 35 ± 6 to 65 ± 8 pA (n = 7, p < 0.05) or from 12 ± 2 to 27 ± 4 pA (n = 7, p < 0.05). Moreover, with continued exposure to 10 μM picaridin, subsequent addition of ranolazine (Ran, 10 μM) markedly attenuated I_Na(P) magnitude measured at −20 or −80 mV to 42 ± 7 (n = 7, p < 0.05) or 17 ± 3 pA (n = 7, p < 0.05), respectively. As such, it was reasonable to assume that the strength of I_Na(P)’s Hys_(V) in response to long-lasting triangular V_ramp was susceptible to being enhanced in the presence of picaridin.

2.7. Picaridin-Mediated Increase of Resurgent I_Na (I_Na(R)) Measured from GH₃ Cells

I_Na(R) has been increasingly demonstrated to be responsible for burst generation and membrane excitability present in different excitable cells [32,41,51,52,53,54,55,56,57,58]. In this regard, we, thus, next wanted to study if the existence of picaridin produced any effects on I_Na(R) identified from these cells. The experiments used to measure I_Na(R) were, then, undertaken in situations where the examined cell was maintained at −80 mV and a brief depolarizing step to +30 mV for a duration of 30 ms was given to activate I_Na(T). Following the abrupt depolarizing step, a series of down-sloping V_ramp’s from +30 to −80 mV, with varying durations in a geometric progression (with a common ratio of 2), was subsequently given to evoke I_Na(R) (Figure 7) [54]. The I_Na(R) amplitude evoked by such down-sloping V_ramp became progressively increased as cells were exposed to picaridin. For example, as GH₃ cells were exposed to picaridin at a concentration of 3 or 10 μM, the I_Na(R) amplitude at the level of −50 mV evoked by an 8.2-s down-sloping V_ramp was respectively raised to 49.8 ± 3.3 pA (n = 7, p < 0.05) or 53.3 ± 3.5 pA (n = 7, p < 0.05) from a control value of 46.1 ± 3.1 pA (n = 7). Moreover, under continued exposure to picaridin, subsequent addition of Ran (10 μM) noticeably attenuated I_Na(R) amplitude at the same level of membrane potential to 46.6 ± 3.2 pA (n = 7).

2.8. Molecular Docking on Interaction between Na_V1.7 Channel and Picaridin

In this study, we also continued to explore how the protein of the hNa_V1.7 channel was optimally docked with the picaridin molecule, by using PyRx software. The protein structure of hNa_V1.7 was taken from RCSB PDB (PDB ID: 5EK0) [59]. The predicted docking sites of the picaridin molecule, with which the amino-acid residues can interact, are presented in Figure 8A,B. The picaridin molecule was noted to form hydrophobic contacts with certain amino-acid residues, such as Val1501(D), Phe1506(D), Ile 1510(D), Glu1550(D), Asp1565(D), Arg1611(D), and Ala1615(D). The atoms in the picaridin molecule also have several hydrogen bonds with residue Thr1507(D) at a distance of 3.13 Å, Arg 1554(D) at 3.01 Å, Ser1568(D) at 3.16 Å, and Arg1620(D) at 3.25 Å, respectively (Figure 8A). The results of molecular docking hinted at the possibility that, apart from the ability of this compound to be active in targeting the odorant binding protein 1 (OBP1) [20,22], the picaridin molecule could potentially dock to the transmembrane region (position: 1597–1613) of hNa1.7 channel (PDB: 5EK0) with a binding affinity of −5.2 kcal/mol.

3. Discussion

In the present work, we provided evidence to show that the presence of picaridin, known to exert a repellent effect on insects, was able to exert stimulatory action on I_Na(T) and I_Na(L) seen in pituitary GH₃ cells. The I_Na(L) in response to brief step depolarization was stimulated to a greater extent than the I_Na(T). Consequently, effective EC₅₀ values needed for picaridin-stimulated I_Na(T) and I_Na(L) in these cells were yielded as 32.7 and 2.8 μM, respectively. The overall I–V relationship of I_Na(T) was shifted to a hyperpolarized potential by approximately 20 mV during exposure to picaridin, whereas the quasi-steady-state inactivation curve of I_Na(T) was shifted to a more depolarized potential by about 5 mV, being devoid of changes in the gating charge of the curve in its presence. The decaying time course of I_Na(T) activated during pulse train stimulation became noticeably slowed in the presence of this compound. Moreover, further addition of brivaracetam (BRV) attenuated picaridin-induced increase of I_Na(T)’s decaying time constant responding to rapid membrane depolarizations. The recovery time course of I_Na(T) during the preceding conditioning pulse train was also strikingly altered upon cell exposure to this agent. When cells were exposed to this agent, the Hys_(V) magnitude in response to the upright isosceles-triangular V_ramp became strikingly augmented. The I_Na(R) magnitude in response to the descending V_ramp following initial brief depolarization was greatly enhanced by adding picaridin. Together, the activity of Na_V channels inherent in excitable cells (e.g., GH₃ cells) may confer susceptibility to perturbations by picaridin or other structurally similar compounds; although, unlike other piperidine derivatives [28,30,32], this agent was observed to induce an excitatory action on I_Na. The differential stimulation by picaridin of I_Na(T) and I_Na(L) is important and it may, thus, be responsible for its modulation of the electrical behaviors of excitable cells [3,60,61], presuming that the findings appear in vivo.

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. Therefore, it is reasonable to assume that the existence of picaridin results in a gain-of-function change perturbed by the slowed time course of I_Na(T) inactivation, and that picaridin-induced increase of measured I_Na(T) is concurrently associated with conceivable use-dependent attenuation of I_Na(T), elicited as either high-frequency firing of action potentials or rapid repetitive stimuli occurring [38,42,62,63,64,65,66].

In this work, we also revealed that GH₃-cell exposure to picaridin could cause the modifications on the recovery of I_Na(T) inactivation evoked by a train of conditioning depolarizing stimuli. Of note, under our experimental observations, the inactivation recovery process of measured I_Na(T), following the conditioning membrane depolarizations, was found to consist of two different fractions of current recovery, i.e., fast and slow recovering pool of I_Na(T). In other words, in agreement with earlier observations [38,41,44,67], the I_Na(T) activated by a conditioning train of pulses can lead to a certain fraction of Na_V channels to shift toward the slowly recovering pool during the current recovery process. As a result, there appears to be two distinct fractions of recovering pool (i.e., fast and slow) following the conditioning rapid membrane depolarization [38,68]. Furthermore, as summarized in Table 1, upon continued exposure to picaridin, the fraction of the fast recovery pool of Na_V channels observed under these experimental conditions tended to become greater, while that in the slow recovery pool of the channel was smaller.

Work in our laboratory explicitly demonstrated the non-equilibrium Hys_(V) behavior of I_Na(P) activated by the upright isosceles-triangular V_ramp, strongly indicating the striking voltage dependence of double V_ramp-evoked I_Na(P) [49]. The experimental results also revealed two distinct types of measured Hys_(V) loops, which is reminiscent of the dynamics of the Lorenz-like system (i.e., figure-of-eight configuration) [69]. In other words, the Hys_(V) motion of the current as a function of time was noted to move in both counterclockwise and clockwise directions. One is a high-threshold counterclockwise loop with a peak of −20 mV, while the other is a low-threshold clockwise loop with a peak falling at around −80 mV. Furthermore, the exposure to picaridin was noted to accentuate Hys_(V)’s strength of I_Na(P) responding to triangular V_ramp. In continued exposure to picaridin, further addition of ranolazine markedly resulted in an attenuation of picaridin-mediated increase of Hys_(V) strength seen in GH₃ cells. Ranolazine has been previously demonstrated to be an inhibitor of I_Na(L) [28,29,33,34]. Findings from the current observations, therefore, unveiled that the double V_ramp-induced I_Na(P) resulted in striking Hys_(V) changes in voltage dependence and that such strength can be evidently enhanced by adding picaridin.

In our study, the I_Na(R) in response to varying durations of repolarizing voltage was robustly detectable [32,41]. Of additional note, when cells were continually exposed to picaridin, the magnitude of I_Na(R) became overly increased. Moreover, the subsequent addition of ranolazine, still during continued exposure to picaridin, was able to attenuate the I_Na(R) magnitude, especially at the level of −50 mV. In accordance with previous reports showing that the I_Na(R) magnitude may alter the I_Na(T) inactivation during high-frequency stimulation [32,51,52,53,55,56,57,58,63,68], the current investigations allowed us to indicate that the existence of picaridin may perturb the electrical behaviors (e.g., aberrant and rapid firing of action potentials) residing in varying excitable cells.

4. Materials and Methods

4.1. Chemicals, Drugs, REAGENTS and Solutions Used in This Work

Zingerone Picaridin (icaridin, KBR 3023, Bayrepel^TM, saltidin^®_, 1-piperidinecarboxylic acid 2-(2-hydroxyethyl)-1-methylpropylester, hydroxyethyl isobutyl piperidine carboxylate [INCI], butan-2-yl 2-(2-hydroxyethyl)piperidine-1-carboxylate [IUPAC], C₁₂H₂₃NO₃) were acquired from MedChemExpress (MCE^®; Everything Biotech, New Taipei City, Taiwan). Ranolazine (Ran), tefluthrin, tetraethylammonium chloride (TEA), and tetrodotoxin (TTX) were obtained from Sigma-Aldrich (Genechain, Kaohsiung, Taiwan), while brivaracetam (BRV) was kindly provided by UCB (Union Chimique Belge) Pharm (PRA Health Sciences, Taipei, Taiwan). To protect picaridin from being degraded by light, stock solution containing this compound was wrapped with aluminum foil, and it was then kept under −20 °C for long-term storage. Unless specified otherwise, fetal calf serum, horse serum, culture media (e.g., Ham’s F-12 medium), L-glutamine, and trypsin/EDTA were mostly acquired from HyClone^TM (Genechain), whereas other chemicals or reagents were acquired from Sigma-Aldrich or Merck (Genchain) and were of laboratory grade.

The standard external solution (i.e., normal Tyrode’s solution) had the composition (in mM): NaCl 136.5, CaCl₂ 1.8, KCl 5.4, MgCl₂ 0.53, glucose 5.5, HEPES 5.5, and the pH was adjusted to 7.4 by adding NaOH. To record ionic flowing through K⁺ currents, the electrode was filled up with the internal pipette solution containing (in mM): K-aspartate 130, KCl 20, KH₂PO₄ 1, MgCl₂ 1, EGTA 0.1, Na₂ATP₃, Na₂GTP 0.1, and HEPES 5, and the pH was adjusted to 7.2 by adding KOH. However, to measure I_Na, K⁺ ions in the pipette solution were replaced with equimolar Cs⁺ ions, and the pH was adjusted to 7.2 with CsOH. The twice-distilled water used for the current experiments was deionized through Millipore-Q^® system (Merck, Tainan, Taiwan).

4.2. Cell Preparations

Clonal pituitary (GH₃) somatolactotrophs, originally acquired from the Bioresources Collection and Research Center ([BCRC-60015, http://catalog.bcrc.firdi.org.tw/BcrcContent?bid=60015] (accessed on 18 August 2022), Hsinchu, Taiwan), were cultured in Ham’s F-12 medium, which was supplemented with 15% heat-inactivated horse serum (v/v), 2.5% fetal calf serum (v/v), and 2 mM L-glutamine. They were grown at 37 °C in monolayer cultures in 50-mL plastic culture flasks in a humidified environment of 5% CO₂/95% air. We performed electrical recordings 5 or 6 days after GH₃ cells underwent subculture (60–70% confluence).

4.3. Electrophysiological Measurements

Shortly before the measurements, we carefully dispersed cells by using 1% trypsin-EDTA solution, and quickly put a small portion of the suspension containing clumps of cells into a recording cubicle fixed on the stage of an inverted microscope (DM-IL; Leica; Major Instruments, Tainan, Taiwan). We maintained cells at room temperature (20–25 °C) in HEPES-buffered normal Tyrode’s solution, the ionic compositions of which are stated above. The patch electrodes used to record were constructed from melting point capillaries made of Kimax^®-34500-99 glass with 1.5–1.8 mm outer diameter (Sigma-Aldrich, Genechain, Kaohsiung, Taiwan) by using a PP-83 vertical puller (Narishige, Major Instruments). When filled with different internal solutions described above, the recording pipettes had tip resistances of 3–5 MΩ. The electrodes were held in place by micromanipulators. We recorded ionic currents in the whole-cell configuration by making some small modifications to the original patch-clamp technique with an RK-400 patch amplifier (Bio-Logic, Claix, France), as described elsewhere [31,32,70,71]. The GΩ-seals, which were made in an all-or-nothing fashion, led to a significant improvement in the signal-to-noise ratio. The liquid junction potential, which occurred due to different compositions between external and internal solutions, became nulled shortly before the GΩ-seal formation was made, and the whole-cell data were then corrected.

4.4. Data Recordings and Processing

The signal output (i.e., potential and current traces) was monitored and digitized online at 10 kHz or more in an ASUS ExpertBook laptop computer (Yuan-Dai, Tainan, Taiwan). For analog-to-digital (A/D) and digital-to-analog (D/A) conversion, a Digidata^® 1440 A device interfaced to the computer was controlled by pCLAMP^TM 10.6 program run under Microsoft Windows 7 (Redmond, WA, USA) [70,71]. We low-pass filtered current signals at 2 kHz by using a FL-4 four-pole Bessel filter (Dagan, Minneapolis, MN, USA). The voltage-clamp protocols, consisting of manifold rectangular or ramp waveforms, were specifically designed, and they were then delivered to the tested cells through D/A conversion.

4.5. Data Analyses for Whole-CELL Ionic Currents

To determine concentration-dependent stimulation of picaridin on the amplitude of I_Na(T) or I_Na(L), we placed GH₃ cells into Ca²⁺-free Tyrode’s solution. During the measurements, we maintained each cell at −100 mV, and a short depolarizing pulse to −10 mV for 30 ms was delivered to evoke I_Na. The I_Na(T) or I_Na(L) magnitudes were then, respectively, measured at the beginning- or end-pulse of 30-ms depolarizing step when cumulative application of different picaridin concentrations was given. The amplitude of I_Na(T) obtained in the presence of picaridin at a concentration of 100 μM was taken to be 100% and current magnitudes during exposure to varying concentrations of this compound were subsequently compared. The concentration-dependent stimulation by picaridin of I_Na(T) or I_Na(L) residing in GH₃ cells was determined by fitting experimental data set to a modified Hill function [31,32]. That is:

$$percentage increase (\%)=\left(E_{max}\times {\left[picaridin\right]}^{n_H}\right)/\left(E{C}_{50}^{n_H}+{\left[picaridin\right]}^{n_H}\right)$$

where [picaridin] = the picaridin concentration given; n_H = the Hill coefficient (i.e., coefficient for cooperativity); EC₅₀ = the concentration required for a 50% stimulation of I_Na(T) or I_Na(L) amplitude activated by abrupt depolarizing step; and E_max = maximal stimulation of I_Na(T) or I_Na(L) caused by the picaridin presence.

To evaluate picaridin-mediated adjustments on the quasi-steady-state inactivation of I_Na(T), the relationship of the normalized amplitude of I_Na(T) versus the conditioning potential obtained with or without the presence of 10 μM picaridin was established, and the data set was then fitted optimally to a Boltzmann function on the following form:

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

where I_max = the maximal I_Na(T) magnitude acquired in the absence or presence of picaridin (10 μM) at the conditioning membrane potential of −100 mV; V = the conditioning potential; V_1/2 = the potential required for half-maximal activation (i.e., midpoint potential); q = the apparent gating charge needed; F = Faraday’s constant; R = the universal gas constant; T = the absolute temperature; and RT/F = 25.2 mV.

By use of a two-step voltage protocol with variable interpule intervals in a geometric progression, the recovery time course of I_Na from block activated in response to the 1-s conditioning pulse train was optimally constructed, and the results acquired with or without the addition of picaridin were thereafter drawn by plotting the I_Na(T) amplitude (normalized with respect to the steady-state amplitude activated at 0.1 Hz). A basic assumption of the analyses was that the recovery time course of the current measured under these experimental conditions could be described in a satisfactory manner by exponential function [38,67,72]. Due to the recovery time course in GH₃ cells exhibiting a rapidly rising recovery phase, followed by a late slow phase, there should be at least two underlying exponential terms. Therefore, the data points showing recovery time course with or without exposure to picaridin were least-squares fitted to the exponential function with biexponential process. That is:

$$y=A\times \left(1-e^{\raisebox{1ex}{$-t$}\!\left/ \!\raisebox{-1ex}{${\tau }_{fast}$}\right.}\right)+B\times \left(1-e^{\raisebox{1ex}{$-t$}\!\left/ \!\raisebox{-1ex}{${\tau }_{slow}$}\right.}\right)$$

(1)

where y is the relative I_Na(T) amplitude at time t, A or B is the relative amplitude of each exponential component, and τ_fast or τ_slow is the fast or slow time constant in the recovery of I_Na block, respectively. The experimental results were optimally analyzed and they are summarized in Table 1.

4.6. Curve-Fitting Approximations and Statistical Analyse

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). The size of independent observations (n) is indicated in cell numbers collected during the experiments. The data distribution obtained was found to satisfy the tests for normality. For comparisons between the two different groups, we performed Student’s t-tests (for paired or unpaired samples). However, for more than two different groups, analysis of variance (for one- or two-way ANOVA) with or without repeated measure was followed by a post-hoc Fisher’s least-significance difference test, which was applied where appropriate to evaluate individual differences among multiple groups of data. A statistical significance (indicated with *, ** or + in the figures) was considered when p < 0.05.

5. Conclusions

The emerging data reveal that picaridin, known to be an insect and acarid repellent in the piperidine chemical family, leads to profound stimulation in I_Na in concentration-, time-, voltage-, frequency-, and Hys_(V)-dependent manners. The precise ionic mechanism of picaridin actions on I_Na needs to be further clarified. The GH₃ cell line used was demonstrated to express the α-subunits of Na_V1.1, Na_V1.2, Na_V1.3, and Na_V1.6, as well as the β1 and β3-subunits of Na_V channel [24,73]. Moreover, the results of molecular docking also prompt us to predict the effectiveness of picaridin in binding to certain amino-acid residues intrinsically in the hNa_V1.7 channel, by forming either hydrophobic contacts or hydrogen bonds. The present data obtained from GH₃ cells, thus, sheds light on the evidence demonstrating the effectiveness of picaridin in adjusting specific ionic currents (e.g., I_Na(T), I_Na(L), I_Na(P) and I_Na(R)). These results may be linked to the additional, but important, actions of picaridin, or other structurally similar compounds, which are potentially useful in pharmacological or toxicological applications in different excitable cells occurring in vivo.