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

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 [INa], has not been entirely explored. In GH3 pituitary tumor cells, we demonstrated that with exposure to picaridin the transient (INa(T)) and late (INa(L)) components of voltage-gated Na+ current (INa) 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 INa(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 INa(T) induced during repetitive stimuli became retarded during its exposure. The recovery time course from the INa 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 INa 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 INa (INa(P)), responding to triangular ramp voltage, was also enhanced during cell exposure to picaridin. The magnitude of resurgent INa (INa(R)) was raised in its presence. Picaritin-induced increases of INa(P) or INa(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 hNaV1.7 channel. Taken literally, the stimulation of INa exerted by the exposure to picaridin is expected to exert impacts on the functional activities residing in electrically excitable cells.


Stimulatory Effect of Picaridin on Voltage-Gated Na + Current (I Na ) Experimentally Recorded from Pituitary GH 3 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 Ca 2+ -free Tyrode's solution as a bathing medium in which 10 mM tetraethylammonium chloride (TEA) and 0.5 mM CdCl 2 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., EC 50 ) 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.
The relationship between the picaridin concentration and the percentage increase of INa(T) or INa(L) was further constructed ( Figure 1B). Addition of picaridin was noticed to increase the INa(T) or INa(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 INa(T) or INa(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 INa(T) and INa(L) in a concentration-dependent manner.  , red open circles) or sustained (late, I Na(L) , blue filled circles) caused by picaridin. The amplitude of I Na(T) or I Na(L) was taken at the start-or end-pulse of the depolarizing command voltage from −100 to −10 mV for a duration of 30 ms. Each point represents the mean ± SEM (n = 8). The sigmoidal curves, on which the data points are overlaid, were reliably fitted to a modified Hill equation, as detailed under Materials and Methods.

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 GH 3 -cell exposure to 10 µM picaridin.
picaridin. The amplitude of INa(T) or INa(L) was taken at the start-or end-pulse of the depolarizing command voltage from −100 to −10 mV for a duration of 30 ms. Each point represents the mean ± SEM (n = 8). The sigmoidal curves, on which the data points are overlaid, were reliably fitted to a modified Hill equation, as detailed under Materials and Methods.

Effect of Picaridin on Mean Current Versus Voltage (I-V) Relationship of INa(T)
We next examined the perturbations of this compound on INa(T) measured at various membrane potentials. As depicted in Figure 2, a steady-state I-V relationship of INa(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 INa(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 INa(T) magnitude from 744 ± 49 to 940 ± 56 pA (n = 7, p < 0.05). Moreover, the steady-state I-V relationship of INa(T) was found to be shifted to more negative potentials during GH3-cell exposure to 10 μM picaridin. The voltage-clamp protocol imposed is shown in the uppermost part. (B) Steady-state I-V relationship of INa(T) without (blue filled squares) and with (red open circles) cell exposure to 10 μM picaridin (mean ± SEM; n = 8 for each point). Note that cell exposure to picaridin depressed the amplitude of INa(T); moreover, the overall steady-state I-V relationship of the current was shifted to more negative potentials in its presence.

Characterization of Picaridin-Induced Modifications on the Steady-State Inactivation Curve of INa(T)
To further characterize the stimulatory effect of picaridin on INa(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 INa(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 Figure 2. Mean current versus voltage (I-V) relationship of I Na(T) caused by picaridin. Current amplitude (i.e., I Na(T) ) was taken at the start of each abrupt depolarization. (A) Exemplar current traces achieved in the control period (upper) and during cell exposure to 10 µM picaridin (lower). The voltage-clamp protocol imposed is shown in the uppermost part. (B) Steady-state I-V relationship of I Na(T) without (blue filled squares) and with (red open circles) cell exposure to 10 µM picaridin (mean ± SEM; n = 8 for each point). Note that cell exposure to picaridin depressed the amplitude of I Na(T) ; moreover, the overall steady-state I-V relationship of the current was shifted to more negative potentials in its presence.

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 GH 3 cells.
of −80 mV. The interval between two sets of voltage pulses applied was about 1 min to allow complete recovery of INa(T). The relationship between the conditioning potentials and the normalized amplitudes of INa(T) with or without the picaridin addition were plotted and, then, appropriately fitted to a Boltzmann function, detailed under Materials and Methods. In control, V1/2 = −48 ± 2 mV, q = 3.5 ± 0.6 e (n = 7), whereas in the presence of 10 μM picaridin, V1/2 −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 INa(T), but it also shifted the steady-state inactivation curve of INa(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 INa(T) magnitude in a voltagedependent fashion in GH3 cells.

Picaridin-Induced Retardation in Cumulative Inhibition of INa(T) Inactivation during Rapid Depolarizing Stimuli
It has been previously demonstrated that, prior to being activated during repetitieve short pulses, the inactivation of INa(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 INa(T) during a train of membrane depolarizations. The tested cell . Modification by picaridin on steady-state inactivation curve of I Na(T) identified from GH 3 cells. This set of experiments was undertaken in situations where conditioning voltage pulses with a duration of 30 ms to various potentials, ranging between −100 and +30 mV, were applied from a holding potential of −80 mV. Following each conditioning pulse, a test pulse to −10 mV for a duration of 30 ms was, subsequently, applied to evoke I Na(T) . An example of current traces obtained by a two-pulse protocol in the control period (upper) and during the exposure to 10 µM picaridin (lower) is illustrated in (A). The uppermost part is the voltage protocol given. (B) Quasi-steady-state inactivation curves of I Na(T) obtained with the absence (blue filled squares) and presence (red open circles) of 10 µM picaridine (mean ± SEM; n = 7 for each point). The normalized amplitude of I Na(T) (i.e., I/I max ) was constructed against the different levels of conditioning potential, and the sigmoidal curves (blue and red lines) were fitted to the Boltzmann equation (see Materials and Methods).

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 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. 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. 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 INa(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 INa(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 INa(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 INa(T) inactivation during repetitive depolarizations to 41.1 ± 2.6 ms (n = 7, p < 0.05). BRV was recently reported to suppress INa(T) amplitude [43]. Overall, these results meant that, apart from the increase in INa(T) amplitude, with cell exposure to picaridin the decrease in the decaying of INa(T) in response to a 1-s train of depolarizing pulses (i.e., accumulative inactivation of INa(T)) could become retarded in these cells.  and picaridin plus ranolazine (Ran) on the time constant of current decay in response to a train of depolarizing command voltage from −80 to −10 mV (mean ± SEM; n = 7 for each point). Current amplitude was measured at the beginning of each depolarizing pulse during depolarizing stimuli. * Signficantly different from control (p < 0.05), ** Significantly different from picaridin (3 μM) alone group (p < 0.05), and + Significantly different from picaridin (10 μM) alone group (p < 0.05).

Effect of Picaridin on the Recovery Time Course of INa(T) Inactivation after the Conditioning Train of Depolarizing Stimuli
The earlier investigations demonstrated a distinguishing type of recovery process from INa(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 INa(T) could be modified by adding picaridin. In this separate set of experiments, the preceding condition train given was composed of 40 20ms 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 INa(T). That is, the INa(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 INa(T) in response to the conditioning depolarizing stimuli, was noticed to emerge largely in a biphasic manner (i.e., fast and slowly recovering pools), . The gray continuous lines on which the data points are overlaid was accurately fitted by a single exponential. Note that with cell exposure to picaridin, the time course of I Na(T) inactivation during a train of depolarizing pulses slowed, together with an increase in time constant of the current inactivation. (C) Summary graph demonstrating the effect of picaridin (3 or 10 mM) and picaridin plus ranolazine (Ran) on the time constant of current decay in response to a train of depolarizing command voltage from −80 to −10 mV (mean ± SEM; n = 7 for each point). Current amplitude was measured at the beginning of each depolarizing pulse during depolarizing stimuli. * Signficantly different from control (p < 0.05), ** Significantly different from picaridin (3 µM) alone group (p < 0.05), and + Significantly different from picaridin (10 µM) alone group (p < 0.05).

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 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 GH 3 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.  [38,44]. Moreover, of interest, during exposure to picaridin, the fraction of the slowly recovering pool of NaV channels during the conditioning train pulse became smaller, while that of the fast recovery pool of the channels tended to be greater; however, both τfast and τslow 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 Ca 2+ -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.

Picaridin-Mediated Increase of Resurgent INa (INa(R)) Measured from GH3 Cells
INa(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 INa(R) identified from these cells. The experiments used to measure INa(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 INa(T). Following the abrupt Figure 6. Modification by picaridin on voltage-dependent hysteresis (Hys (V) ) of persistent I Na (I N(P) ) elicited by an upright isosceles-triangular ramp voltage (V ramp ). The experiments were undertaken when the tested cell was voltage-clamped at −80 mV, and the V ramp from −100 to +50 mV for a duration of 1.2 s (i.e., ramp speed of ±0.25 mV/ms) was applied to it. (A) Exemplar current traces acquired during the control period (upper part) and in the presence of 10 µM picaridin (lower part). The trajectory shown in blue or red color indicates current trace evoked at the ascending (up-sloping) or descending (down-sloping) limb of the upright triangular V ramp , respectively. The voltage protocol is illustrated atop current traces. The dashed curve arrows in each panel represent the direction of potential or current trajectory by which time goes. Summary graph showing the effects of picaridin (3 or 10 µM) and picaridin plus Ran on I Na(P) amplitude activated by the up-sloping (ascending), (B) or down-sloping (descending), (C) limb of 1.2-s triangular V ramp is, respectively, illustrated. The I Na(P) amplitude at the up-sloping or down-sloping end of V ramp was measured at −20 or −80 mV, respectively. Each point indicates the mean ± SEM (n = 8). * Significantly different from control (p < 0.05), ** Significantly different from picaridin (3 µM) alone group (p < 0.05), and + Significantly different from picaridin (10 µM) alone group (p < 0.05).

Picaridin-Mediated Increase of Resurgent I Na (I Na(R) ) Measured from GH 3 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 GH 3 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).

Molecular Docking on Interaction between NaV1.7 Channel and Picaridin
In this study, we also continued to explore how the protein of the hNaV1.7 channel was optimally docked with the picaridin molecule, by using PyRx software. The protein structure of hNaV1.7 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  . Augmentation by picaridin on resurgent I Na (I Na(R) ) evoked by the descending V ramp with varying durations. The examined cell was held at the level of −80 mV, and a 30 ms depolarizing pulse to +30 mV was imposed. Following brief step depolarization, the down-sloping V ramp from +30 to −80 mV, with varying duration in a geometric progression, was delivered to the cells. (A) In our voltage-clamp protocol, the different colors indicated in current traces (lower) correspond with those in potential traces (upper). Representative current traces obtained without (upper) or with (lower) the exposure to 10 µM picaridin. The uppermost part is the voltage-clamp protocol given, and the arrow indicates the level of −50 mV at which I Na(R) amplitude evoked by V ramp with a duration of 8.2 s was measured. (B) Summary graph demonstrating effects of picaridin (3 or 10 µM) and picaridin plus Ran on I Na(R) amplitude (mean ± SEM; n = 7 for each point). Current amplitude was measured at the level of −50 mV during the 8.2-s descending V ramp . * This result is significantly different from control (p < 0.05), ** Significantly different from picaridin (3 µM) alone group (p < 0.05), and + Significantly different from picaridin (10 µM) alone group (p < 0.05).

Molecular Docking on Interaction between Na V 1.7 Channel and Picaridin
In this study, we also continued to explore how the protein of the hNa V 1.7 channel was optimally docked with the picaridin molecule, by using PyRx software. The protein structure of hNa V 1.7 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

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

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 GH 3 cells. The I Na(L) in response to brief step depolarization was stimulated to a greater extent than the I Na(T) . Consequently, effective EC 50 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., GH 3 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 GH 3 -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 GH 3 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.
The standard external solution (i.e., normal Tyrode's solution) had the composition (in mM): NaCl 136.5, CaCl 2 1.8, KCl 5.4, MgCl 2 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, KH 2 PO 4 1, MgCl 2 1, EGTA 0.1, Na 2 ATP 3 , Na 2 GTP 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).

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-ornothing 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.

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.

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 GH 3 cells into Ca 2+ -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 GH 3 cells was determined by fitting experimental data set to a modified Hill function [31,32]. That is: where [picaridin] = the picaridin concentration given; n H = the Hill coefficient (i.e., coefficient for cooperativity); EC 50 = 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:

RT/qF
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 GH 3 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: 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.

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.

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 GH 3 cell line used was demonstrated to express the α-subunits of Na V 1.1, Na V 1.2, Na V 1.3, and Na V 1.6, 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 V 1.7 channel, by forming either hydrophobic contacts or hydrogen bonds. The present data obtained from GH 3 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.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The original data is available upon reasonable request to the corresponding author.

Acknowledgments:
The authors gratefully acknowledge Hsin-Yen Cho and Meng-Cheng Yu for their contributions to the earlier experiments.

Conflicts of Interest:
No conflicts of interest, financial or otherwise, are declared by the author(s). The content and writing of this paper are solely the responsibility of the authors.

Abbreviations
BRV brivaracetam EC 50 concentration required for half-maximal stimulation Hys (V) voltage-dependent hysteresis I Na(L) late Na + current I Na(T) transient (peak) Na + current Na V channel voltage-gated Na + channel Ran ranolazine SEM standard error of mean TEA tetraethylammonium chloride τ inact(S) slow component in inactivation time constant V ramp ramp voltage TTX tetrodotoxin V ramp ramp voltage