Inhibition of Voltage-Gated Na+ Currents Exerted by KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea), an Inhibitor of Na+-Ca2+ Exchanging Process

KB-R7943, an isothiourea derivative, has been recognized as an inhibitor in the reverse mode of the Na+-Ca2+ exchanging process. This compound was demonstrated to prevent intracellular Na+-dependent Ca2+ uptake in intact cells; however, it is much less effective at preventing extracellular Na+-dependent Ca2+ efflux. Therefore, whether or how this compound may produce any perturbations on other types of ionic currents, particularly on voltage-gated Na+ current (INa), needs to be further studied. In this study, the whole-cell current recordings demonstrated that upon abrupt depolarization in pituitary GH3 cells, the exposure to KB-R7943 concentration-dependently depressed the transient (INa(T)) or late component (INa(L)) of INa with an IC50 value of 11 or 0.9 μM, respectively. Likewise, the dissociation constant for the KB-R7943-mediated block of INa on the basis of a minimum reaction scheme was estimated to be 0.97 μM. The presence of benzamil or amiloride could suppress the INa(L) magnitude. The instantaneous window Na+ current (INa(W)) activated by abrupt ascending ramp voltage (Vramp) was suppressed by adding KB-R7943; however, subsequent addition of deltamethrin or tefluthrin (Tef) effectively reversed KB-R7943-inhibted INa(W). With prolonged duration of depolarizing pulses, the INa(L) amplitude became exponentially decreased; moreover, KB-R7943 diminished INa(L) magnitude. The resurgent Na+ current (INa(R)) evoked by a repolarizing Vramp was also suppressed by adding this compound; moreover, subsequent addition of ranolazine or Tef further diminished or reversed, respectively, its reduction in INa(R) magnitude. The persistent Na+ current (INa(P)) activated by sinusoidal voltage waveform became enhanced by Tef; however, subsequent application of KB-R7943 counteracted Tef-stimulated INa(P). The docking prediction reflected that there seem to be molecular interactions of this molecule with the hNaV1.2 or hNaV1.7 channels. Collectively, this study highlights evidence showing that KB-R7943 has the propensity to perturb the magnitude and gating kinetics of INa (e.g., INa(T), INa(L), INa(W), INa(R), and INa(P)) and that the NaV channels appear to be important targets for the in vivo actions of KB-R7943 or other relevant compounds.


Suppressive Effect of KB-R7943 on Voltage-Gated Na + Current (I Na ) Identified from Pituitary GH 3 Cells
In the initial stage of measurements, we explored if and how the presence of KB-R7943 could produce any perturbations on I Na present in these cells. To prevent any contaminations by either the NCX exchanging process, voltage-gated Ca 2+ currents, or Ca 2+ -induced inward currents [3,4,29,44,45], we placed cells in Ca 2+ -free Tyrode solution, and the measuring pipettes were filled with a solution enriched with Cs + . To evoke I Na , we voltage-clamped each tested cell at −80 mV. A hyperpolarizing pulse to −100 mV for a duration of 30 ms was then applied to precede the depolarizing command voltage from −100 to −10 mV, and such a depolarizing pulse was then given to activate I Na (i.e., I Na(T) and I Na(L) ). Under these experimental conditions, we were able to detect the occurrence of a transient inward current (i.e., inward flux of cations) which displayed the rapidly activating and inactivating time course in the current ( Figure 1A). Upon such a brief rectangular pulse, this type of transient inward current was sensitive to either suppression or stimulation by the presence of tetrodotoxin (TTX, 1 µM) or tefluthrin (Tef, 10 µM), respectively. However, neither cell exposure to nimodipine (1 µM) nor CdCl 2 (0.5 mM) was able to alter the current magnitude. Therefore, it has been identified as a TTX-sensitive I Na [22,28,34]. Furthermore, upon cell exposure to KB-R7943, the transient I Na (I Na(T) ) progressively became diminished in combination with the concurrent increase in inactivation rate of I Na(T) ( Figure 1A). For example, the application of KB-R7943 at a concentration of 1 or 3 µM KB-R7943 considerably decreased I Na(T) amplitude to 887 ± 17 pA (n = 8, p < 0.05) or 693 ± 15 pA (n = 8, p < 0.05), respectively, from a control value of 983 ± 21 pA (n = 8). Concurrently, the time constant (τ inact(S) ) in the slow component of I Na(T) inactivation was accompanied by a significant reduction to 2.1 ± 0.3 ms (n = 8, p < 0.05) or 1.8 ± 0.2 ms (n = 8, p < 0.05), respectively, from a control value of 2.4 ± 0.3 ms (n = 8). However, no considerable changes in the time constant in the fast component of current inactivation were demonstrated with exposure to 1 or 3 µM KB-R7943. After KB-R7943 was removed, I Na(T) amplitude was returned to 979 ± 21 pA (n = 8).
The sigmoidal relationship between the KB-R7943 concentration and the peak (I Na(T) ) or late (I Na(L) ) component of I Na elicited by a short depolarization step from −100 to −10 mV was further constructed. As can be seen in Figure 1B, the cumulative application of KB-R7943 at the concentrations ranging between 0.1 and 30 µM resulted in a concentrationdependent reduction in the magnitude of both I Na(T) and I Na(L) . According to a modified Hill equation described in Section 4, the IC 50 value entailed for KB-R7943-induced suppression of I Na(T) or I Na(L) seen in GH 3 cells was yielded to be 11 or 0.9 µM, respectively. Therefore, the results reflected that with exposure to this compound, the observed I Na(L) magnitude was diminished to a greater extent than I Na(T) . Furthermore, in accordance with IC 50 values, with the minimal reaction scheme stated in the Supplementary Information, the value of dissociation constant (K D ) with the presence of KB-R7943 was calculated to be 0.97 µM, which was close to the IC 50 for its inhibitory action on I Na(L) ; however, the K D value was lower than IC 50 for its suppression on I Na(T) . The experimental observations allowed us to indicate that cell exposure to KB-R7943 concentration-dependently produced an inhibitory but differential effect on the magnitude of I Na(T) and I Na(L) .

Comparison among Effects of Benzamil, Amiloride, Benzamil plus Tefluthrin (Tef), and Benzamil plus Deltamethrin (DLT) on I Na(L) Amplitude Measured from GH 3 Cells
We next explored if several other compounds (e.g., benzamil and amiloride) known to suppress the activity of NCX exchanging process could exert any modifications on the amplitude of I Na in response to rapid membrane depolarization. Either benzamil or amiloride has been previously reported to suppress the activity of the NCX exchanging process, which is also present in pituitary cells [38,46]. Of note, as demonstrated in Figure 2, either further addition of benzamil (10 µM) or amiloride (10 µM) was able to diminish the amplitude of I Na(T) or I Na(L) in combination with a measurable raise in the inactivation time course (i.e., decrease in I Na(T) 's τ inact(S) ). Moreover, with continued exposure to 10 µM benzamil, the subsequent addition of tefluthrin (Tef, 10 µM) or deltamethrin (DLT, 10 µM) was effective at reversing the benzamil-mediated decrease in I Na(L) measured at the endpulse of the short depolarizing step. Tef or DLT, which belongs to pyrethroid insecticides, has been reported earlier to be an activator of I Na [22,29,32,34,36]. Therefore, cell exposure to benzamil or amiloride at a concentration of 10 µM can cause a reduction in I Na(T) and I Na(L) magnitude.  The whole−cell current recordings were conducted in cells bathed in Ca 2+ −free Tyrode solution containing 10 mM tetraethylammonium chloride (TEA) and 0.5 mM CdCl 2 , and we filled up the measuring electrode with a solution containing Cs + . (A) Exemplar current traces obtained during control period (a, black color) and in the presence of 1 µM KB−R7943 (b, pink color, KB) or 3 µM KB−R7943 (c, red color, KB). The upper part shown the voltage clamp protocol given; graphs on the right side of (A) with dashed curve arrows show the expanded records from each purple broken box in the left side. (B) Concentration-response curve of KB−R7943−mediated inhibition of transient (peak) I Na (I Na(T) ) (open red squares) or late (sustained) I Na (I Na(L) ) (filled blue circles) observed in GH 3 cells. The smooth blue or red line drawn represents the goodness-of-fit to the modified Hill equation, as elaborated in Section 4. The IC 50 value for KB-R7943-induced inhibition of I Na(T) or I Na(L) seen in these cells was yielded to be 11 or 0.9 µM, respectively. Each point represents the mean ± SEM (n = 8−9).

Inhibitory Effect of KB-R7943 on Average Steady-State Current Versus Voltage (I-V) Relationship of I Na(T)
In another separate set of measurements, we held the studied cells at −80 mV and then applied various levels of voltage pulses to them, from −90 to +40 mV in 10 mV increments for a duration of 30 ms. Under the experimental voltage protocols, a family of I Na(T) could be robustly elicited and the currents were noticeably manifested by a rapid activating and inactivating property. Of note, one minute after cell exposure to 3 µM KB-R7943, the I Na(T) magnitude became depressed, especially at the potentials ranging between −20 and +20 mV. Figure 3 depicts the I-V relationships (i.e., V-shaped configuration) of I Na(T) measured at the beginning of each potential in the control period (i.e., absence of KB-R7943) and during exposure to 3 µM KB-R7943. The results hence showed that the overall quasi-steady-state I-V relationship of I Na(T) remained unchanged with the presence of KB-R7943, despite its ability to decrease I Na(T) amplitude.
cess, which is also present in pituitary cells [38,46]. Of note, as demonstrated in Figure 2, either further addition of benzamil (10 μM) or amiloride (10 μM) was able to diminish the amplitude of INa(T) or INa(L) in combination with a measurable raise in the inactivation time course (i.e., decrease in INa(T)'s τinact(S)). Moreover, with continued exposure to 10 μM benzamil, the subsequent addition of tefluthrin (Tef, 10 μM) or deltamethrin (DLT, 10 μM) was effective at reversing the benzamil-mediated decrease in INa(L) measured at the endpulse of the short depolarizing step. Tef or DLT, which belongs to pyrethroid insecticides, has been reported earlier to be an activator of INa [22,29,32,34,36]. Therefore, cell exposure to benzamil or amiloride at a concentration of 10 M can cause a reduction in INa(T) and INa(L) magnitude.  Effect of benzamil, amiloride, benzamil plus tefluthrin (Tef), and benzamil plus deltamethrin (DLT) on I Na measured from GH 3 cells. (A) Exemplar current traces elicited by rectangular depolarizing pulse from −80 to −10 mV for a duration of 40 ms. a: control (black color); b: in the presence of 10 µM benzamil (b, red color). The uppermost part is the voltage clamp protocol delivered, and the lower part of (A) shows the display of the expanded records in each purple dashed box. (B) Summary graph demonstrating effects of benzamil, amiloride, benzamil plus tefluthrin (Tef), and benzamil plus deltamethrin (DLT) on the amplitude of I Na(L) (mean ± SEM; n = 7 for each yellow bar). The I Na(L) amplitude was measured at the end of a short depolarizing pulse from −80 to −10 mV for a duration of 40 ms. * Significantly different from control (p < 0.05) and ** significantly different from benzamil (10 µM) alone group (p < 0.05).

Inhibitory Effect of KB-R7943 on Average Steady-State Current Versus Voltage (I-V) Relationship of INa(T)
In another separate set of measurements, we held the studied cells at −80 mV and then applied various levels of voltage pulses to them, from −90 to +40 mV in 10 mV increments for a duration of 30 ms. Under the experimental voltage protocols, a family of INa(T) could be robustly elicited and the currents were noticeably manifested by a rapid activating and inactivating property. Of note, one minute after cell exposure to 3 μM KB-R7943, the INa(T) magnitude became depressed, especially at the potentials ranging between −20 and +20 mV. Figure 3 depicts the I-V relationships (i.e., V-shaped configuration) of INa(T) measured at the beginning of each potential in the control period (i.e., absence of KB-R7943) and during exposure to 3 μM KB-R7943. The results hence showed that the overall quasi-steady-state I-V relationship of INa(T) remained unchanged with the presence of KB-R7943, despite its ability to decrease INa(T) amplitude.

Suppressive Effect of KB-R7943 on the Window Component of I Na (I Na(W) ) Measured from GH 3 Cells
The presence of instantaneous I Na(W) over the short period of time activated in response to the upsloping (or ascending) ramp voltage (V ramp ) has been demonstrated earlier in varying excitable cells [28,29,[47][48][49][50]. We thus proceeded to examine if the KB-R7943 presence could modify the magnitude of non-linear I Na(W) evoked by abrupt ascending V ramp . To perform this separate set of measurements, we held the tested cell at −80 mV, and an ascending V ramp from −100 to +50 mV for a duration of 150 ms (i.e., with a ramp speed of 1 mV/ms) was then imposed to activate instantaneous I Na(W) . Within one minute of exposing GH 3 cells to KB-R7943 (3 or 10 µM), the strength (i.e., ∆area) of I Na(W) induced by the 150 ms upsloping V ramp was profoundly diminished ( Figure 4A,B). For example, treating cells with 10 µM resulted in a striking reduction in I Na(W) 's ∆area from 6.11 ± 0.24 to 1.24 ± 0.15 mV·nA (n = 8, p < 0.05). Moreover, still with continued presence of 10 µM KB-R7943, the subsequent application of either Tef (10 µM) or DLT (10 µM) measurably attenuated the KB-R7943-mediated reduction in ∆area, as demonstrated by a marked elevation of ∆area value to 3.13 ± 0.19 mV·nA (n = 8, p < 0.05) or 3.11 ± 0.19 mV·nA (n = 8, p < 0.05), respectively. It is therefore clear from electrical recordings that the I Na(W) 's strength activated by the ascending V ramp can be subject to being inhibited by the presence of KB-R7943.   of I Na(W) (mean ± SEM; n = 8 for each green bar). The ∆area of I Na(W) (i.e., the relationship of membrane voltage versus current amplitude) was calculated at the area encircled under the voltages ranging between −90 and +40 mV during the short upsloping V ramp . * Significantly different from control (p < 0.05), ** significantly different from KB-R7943 (3 µM) alone group (p < 0.05), and + significantly different from KB−R7943 (10 µM) alone group (p < 0.05).
2.5. KB-R7943-Mediated Slowing in Recovery from I Na(L) either during Prolonged Duration of Depolarizing Pulse or by the Envelope-of-Tail Test As the pulse duration applied for the elicitation of the inward current became prolonged, the NCX exchanging current seen in bullfrog atrial cells was noticed to be overly enhanced [1,3]. We thus continued to investigate if the presence of KB-R7943 could lead to any modifications in recovery from the decay of I Na(L) . The recovery from the current block was conducted with a two-step voltage clamp protocol in situations where the interval of depolarizing command pulses (i.e., conditioning pulse) was progressively prolonged. After each conditioning pulse, a clamp step repolarized back to the level of −50 mV for 20 ms was applied to evoke deactivating I Na ( Figure 5A). As demonstrated in Figure 5B, the relationship between the duration of conditioning pulse and amplitude of deactivating I Na at the end of −50 mV with or without cell exposure to 3 µM KB-R7943 was afterwards established. Of note, the decaying time course of deactivating I Na(L) became slowed with the presence of 3 µM KB-R7943, as demonstrated by a lengthening in decaying time constant of the current from 37 ± 3 to 48 ± 4 ms (n = 7, p < 0.05). Similarly, the relationship of the relative amplitude (i.e., I Na(L) amplitude at −50 mV was divided by that at −10 mV) versus pulse duration (i.e., the envelope-of-tail test for I Na(L) ) noticeably became decayed in an exponential fashion and is constructed in Figure 5C. Similarly, upon exposure to 3 µM KB-R7943, I Na(L) evoked by the envelope-of-tail test was gradually decreased, as evidenced by an increase in decaying time constant of relative amplitude from 34 ± 3 to 48 ± 4 ms (n = 7, p < 0.05). The experimental results led us to reflect that the KB-R7943-mediated decrease in I Na(L) appears to be independent of its suppressive actions on the activity of the NCX exchanging process.

Modification of Nonlinear Resurgent Na + Current (I Na(R) ) in Response to the Descending V ramp
The I Na(R) has been identified in GH 3 cells [27,34], and the magnitude of this current was also previously demonstrated to be intimately associated with high-frequency firing inherent in cerebellar Purkinje neurons [51][52][53][54]. This type of Na + current is particularly unique, because it is not detectable until the membrane potential becomes repolarized below 0 mV. Alternatively, in addition to being activated by depolarizing voltage pulses rather than by repolarizing voltage steps, I Na(R) was found to activate and decay more slowly than I Na(T) [55]. As a result, I Na(R) has been thought to help produce rapid depolarization immediately following an action potential; hence, it is suited either for cells that fire spontaneously at a higher firing rate, or to offer noise modulation in neurons with varying bursting firing [52,54,56]. In this regard, efforts were additionally made to see if the presence of KB-R7943 could exert any perturbations on such an instantaneous current evoked by the descending V ramp . As the whole-cell configuration was firmly made, we imposed the 30 ms depolarizing step from −80 to +30 mV followed by a descending (or repolarizing) V ramp to −80 mV on the tested cell for a duration of 1 s. As demonstrated in Figure 6, the I Na(R) magnitude in response to such voltage clamp protocol became overly reduced during cell exposure to KB-R7943. For example, within one minute of exposing cells to KB-R7943 at a concentration of 1 or 3 µM KB-R7943, I Na(R) amplitude measured at −5 mV decreased from 42.3 ± 2.3 pA (n = 7) to 31.4 ± 2.1 pA (n = 7, p < 0.05) or 19.6 ± 1.5 pA (n = 7, p < 0.05), respectively. However, no clear modification in the voltage level (i.e., around −5 mV) for peak I Na(R) elicitation was demonstrated with the presence of KB-R7943. Moreover, with continued exposure to 3 µM KB-R7943, the subsequent addition of ranolazine (10 µM, Ran) or Tef (10 µM) diminished or increased current amplitude to 3.2 ± 0.9 pA (n = 7, p < 0.05) or 29.4 ± 2.4 pA (n = 7, p < 0.05), respectively. Ran was earlier reported to be an inhibitor of I Na(L) [25,37]. However, neither subsequent application of nimodipine (1 µM) nor CdCl 2 (0.5 mM) had any effects on the KB-R7943-mediated decrease in I Na(R) . It can be interpreted to mean, therefore, that the exposure to KB-R7943 is capable of suppressing I Na(R) magnitude during the descending V ramp observed in these cells.  ranolazine (10 μM, Ran) or Tef (10 μM) diminished or increased current amplitude to 3.2 ± 0.9 pA (n = 7, p < 0.05) or 29.4 ± 2.4 pA (n = 7, p < 0.05), respectively. Ran was earlier reported to be an inhibitor of INa(L) [25,37]. However, neither subsequent application of nimodipine (1 μM) nor CdCl2 (0.5 mM) had any effects on the KB-R7943-mediated decrease in INa(R). It can be interpreted to mean, therefore, that the exposure to KB-R7943 is capable of suppressing INa(R) magnitude during the descending Vramp observed in these cells.

KB-R7943-Mediated Effect on Persistent Na + Current (I Na(P) ) Evoked by Sinusoidal Voltage Waveform
There is growing evidence to show that a significant fraction of subthreshold or background Na + currents is functionally active in varying types of excitable cells [20,22,24,27,[57][58][59][60][61]. Recent investigations have also demonstrated possible modifications of sinusoidal voltage wave on membrane ionic currents [62][63][64][65]. For these reasons, efforts were further given to answer the question of whether I Na(P) can be susceptible to adjustments by sinusoidal voltage waveform or whether or how sinusoidal voltage-induced I Na(P) can be perturbed by adding KB-R7943. An example of a KB-R7943-mediated effect on sinusoidal waveformactivated I Na(P) (transient inward deflection indicated in asterisk) seen in GH 3 cells is illustrated in Figure 7A. It needs to be mentioned that with cell exposure to Tef (10 µM), the difference (i.e., ∆amplitude) taken between current amplitude taken at −60 mV and that at −30 mV was effectively enhanced. Moreover, the subsequent application of KB-R7943 (1 or 3 µM), still in the presence of Tef, could attenuate its stimulation of I Na(P) activated by sinusoidal voltage waveform ( Figure 7B).

Docking Prediction of hNa V 1.7 and KB-R7943
In this study, we further explored how the protein of the hNa V 1.7 channel could be appropriately docked with KB-R7943 with the help of PyRx software. The protein structure of hNa V 1.7 was derived from RCB PDB (ID: 5EK0). The predicted docking sites of the KB-R7943 molecule with which the amino acid residues can interact are presented in Figure 8. Accordingly, it is important to mention that the KB-R7943 molecule may form hydrophobic contacts with certain amino acid residues, including Thr1678(C), Thr1678(D), Leu1679(A), Leu1679(B), Leu1679(C), Leu1679(D), Glu1680(A), Glu1680(B), and Glu1680(D). The atom in the KB-R7943 molecule also has the formation of hydrogen bonds with residue Met1677(C) or Thr1709(C) with an estimated distance of 3.06 and 3.14 Å or 2.92 Å, respectively, and has the formation of hydrogen bonds with residue Ser1681(B) or Ser1681(C) with a distance of 3.02 Å or 2.98 and 2.88 Å, respectively. Therefore, based on the Na V 1.7 protein sequence (GenBank: ASY-04966.1), the inactivation gate of the channel was found to be located at the residue positions ranging between 1459 and 1462, which are noticeably adjacent to the docking sites of the KB-R7943 channel. The results regarding molecular docking thus enable us to propose that the KB-R7943 molecule can appropriately dock to the transmembrane segment (position: 1665-683) of the hNa V 1.7 channel (PDB: 5EK0). Moreover, the binding affinity for molecular docking was estimated to be −7.7 kcal/mol. As a result, in combination with the electrophysiological results described above, such molecular docking might support the notion that KB-R7943 can have a substantial impact on the magnitude and/or gating kinetics of I Na .

KB-R7943-Mediated Effect on Persistent Na + Current (INa(P)) Evoked by Sinusoidal Voltage Waveform
There is growing evidence to show that a significant fraction of subthreshold or background Na + currents is functionally active in varying types of excitable cells [20,22,24,27,[57][58][59][60][61]. Recent investigations have also demonstrated possible modifications of sinusoidal voltage wave on membrane ionic currents [62][63][64][65]. For these reasons, efforts were further given to answer the question of whether INa(P) can be susceptible to adjustments by sinusoidal voltage waveform or whether or how sinusoidal voltage-induced Figure 7A. It needs to be mentioned that with cell exposure to Tef (10 μM), the difference (i.e., ∆amplitude) taken between current amplitude taken at −60 mV and that at −30 mV was effectively enhanced. Moreover, the subsequent application of KB-R7943 (1 or 3 μM), still in the presence of Tef, could attenuate its stimulation of INa(P) activated by sinusoidal voltage waveform ( Figure 7B).  1 or 3 µM) on ∆current amplitude of I Na(P) activated in response to sinusoidal voltage command (mean ± SEM; n = 7 for each brown bar). The absolute value of ∆current amplitude of I Na(P) shown on the y-axis was measured when the difference between current amplitude at −60 mV and that at −30 mV was taken. * Significantly different from control (p < 0.05), ** significantly different from Tef (10 µM) alone group (p < 0.05), + significantly different from Tef (10 µM) plus KB (1 µM) group (p < 0.05).

Docking Prediction of hNa V 1.2 and KB-R7943
It has been previously demonstrated that pituitary GH 3 cells could express the mRNA transcripts for the α-subunit of Na V 1.1, Na V 1.2, Na V 1.3, and Na V 1.6, as well as β1and β3subunits of Na V channels [19]. Therefore, we further investigated how the protein of hNa V 1.2 could be docked by KB-R7943 with PyRx software. The docking sites of the KB-R7943 molecule were shown in Figure 9. Notably, as it is docked to hNa V 1.2, KB-R7943 can form hydrogen bond with residues Glu1788(A) and Thr1862(A) with distances of 2.81 and 2.88 Å, respectively. Furthermore, KB-R7943 can form hydrophobic contacts with several residues, including Glu1788(A), Leu1790(A), Phe1859(A), Lys1863(A), Leu1866(A), Gly1867(A), Glu1871(A), and Leu1875(A). This prediction thus reflects that KB-R7943 can bind to the amino acid residues of the hNa V 1.2 channel with an estimated binding affinity of −6.3 kcal/mol. Such predicted interactions could potentially affect the KB-R7943-mediated change in I Na described above.
drophobic contacts with certain amino acid residues, including Thr1678(C), Thr1678(D), Leu1679(A), Leu1679(B), Leu1679(C), Leu1679(D), Glu1680(A), Glu1680(B), and Glu1680(D). The atom in the KB-R7943 molecule also has the formation of hydrogen bonds with residue Met1677(C) or Thr1709(C) with an estimated distance of 3.06 and 3.14 Å or 2.92 Å , respectively, and has the formation of hydrogen bonds with residue Ser1681(B) or Ser1681(C) with a distance of 3.02 Å or 2.98 and 2.88 Å , respectively. Therefore, based on the NaV1.7 protein sequence (GenBank: ASY-04966.1), the inactivation gate of the channel was found to be located at the residue positions ranging between 1459 and 1462, which are noticeably adjacent to the docking sites of the KB-R7943 channel. The results regarding molecular docking thus enable us to propose that the KB-R7943 molecule can appropriately dock to the transmembrane segment (position: 1665-683) of the hNaV1.7 channel (PDB: 5EK0). Moreover, the binding affinity for molecular docking was estimated to be −7.7 kcal/mol. As a result, in combination with the electrophysiological results described above, such molecular docking might support the notion that KB-R7943 can have a substantial impact on the magnitude and/or gating kinetics of INa.

Docking Prediction of hNaV1.2 and KB-R7943
It has been previously demonstrated that pituitary GH3 cells could express the mRNA transcripts for the -subunit of NaV1.1, NaV1.2, NaV1.3, and NaV1.6, as well as 1and 3subunits of NaV channels [19]. Therefore, we further investigated how the protein of hNaV1.2 could be docked by KB-R7943 with PyRx software. The docking sites of the KB-R7943 molecule were shown in Figure 9. Notably, as it is docked to hNaV1.  The chemical structure of KB-R7943 was taken from PubChem (compound CID: 9823846), whereas the protein structure of hNaV1.2 was from RCB PDB (ID: 2KAV). We docked the KB-R7943 molecule to hNaV1.2 with PyRx, and diagram of the interaction between the hNaV1.2 channel and the KB-R7943 molecule was then generated from LigPlot + . Similar to those on the right side of Figure 8, the red arcs on which red small bars are faced and radiated toward the KB-R7943 molecule represent the hydrophobic interactions, whereas green dotted lines in amino acid residue (i.e., Glu1788(A) and Thr1862(A)) indicate the formation of hydrogen bonds.

Discussion
The important findings in this study are as follows. (a) In pituitary GH3 lactotrophs, the presence of KB-R7943, thought to suppress the activity of the NCX exchanging process, could suppress INa in concentration-, time-, and voltage-dependent manners. (b) The Figure 9. Molecular docking of the hNa V 1.2 channel and the KB-R7943 molecule. The chemical structure of KB-R7943 was taken from PubChem (compound CID: 9823846), whereas the protein structure of hNa V 1.2 was from RCB PDB (ID: 2KAV). We docked the KB-R7943 molecule to hNa V 1.2 with PyRx, and diagram of the interaction between the hNa V 1.2 channel and the KB-R7943 molecule was then generated from LigPlot + . Similar to those on the right side of Figure 8, the red arcs on which red small bars are faced and radiated toward the KB-R7943 molecule represent the hydrophobic interactions, whereas green dotted lines in amino acid residue (i.e., Glu1788(A) and Thr1862(A)) indicate the formation of hydrogen bonds.

Discussion
The important findings in this study are as follows. (a) In pituitary GH 3 lactotrophs, the presence of KB-R7943, thought to suppress the activity of the NCX exchanging process, could suppress I Na in concentration-, time-, and voltage-dependent manners. (b) The estimated IC 50 values required for KB-R7943-inhibited the amplitude of I Na(T) and I Na(L) were distinguishable (i.e., 11 and 0.9 µM, respectively). (c) Either benzamil or amiloride, also known to be inhibitors of the NCX exchanging current, was found to suppress I Na(L) amplitude. (d) The steady-state I-V relationship of I Na(T) remained unaltered in the KB-R7943 presence. (e) The strength (i.e., ∆area) of instantaneous I Na(W) activated by abrupt ascending V ramp became depressed by adding KB-R7943; however, with continued exposure to this compound, further addition of deltamethrin (DLT) or tefluthrin (Tef) effectively attenuated the KB-R7943-mediated decrease in V ramp -induced I Na(W) . (f) As the duration of depolarizing pulse was prolonged, the amplitude of I Na(L) became diminished as a function of time in an exponential fashion; furthermore, the exposure to KB-R7943 decreased I Na(L) amplitude. (g) The presence of this compound suppressed resurgent Na + (I Na(R) ) evoked by the repolarizing V ramp ; moreover, further exposure either to ranolazine (Ran) or Tef, respectively, diminished or attenuated the KB-R7943-mediated decrease in I Na(R) . (h) The persistent I Na (I N(P) ) evoked by sinusoidal voltage waveform was increased by adding Tef; moreover, the subsequent application of KB-R7943 could attenuate such Tef-stimulated I Na(P) . (i) The molecular docking of KB-R7943 to hNa V 1.2 or hNa V 1.7 was predicted because of the presumed formation of both hydrophobic contacts and hydrogen bonds. Taken together, the experimental results therefore allow us to reflect that, in concert with the inhibitory effect on the reverse mode of the NCX exchanging process in varying cell types [6,7,40,46,61,[66][67][68][69], KB-R7943-mediated perturbations in I Na(T) , I Na(L) , I Na(W) , I Na(R) , and I Na(P) tend to be independent of KB-R7943's suppressive action on the activity of the NCX exchanging process. Therefore, these actions are anticipated to participate in potential modifications of the functional activities (e.g., various firing patterns) in electrically excitable cells.
In this study, cell exposure to KB-R7943 was capable of suppressing the amplitude of I Na(T) as well as shortening the inactivation time course of the current activated by abrupt depolarizing voltage command. A concentration-dependent inhibition of I Na(T) or I Na(L) , with effective IC 50 values of 11 or 0.9 µM, respectively, was also obtained. The K D value evaluated from quantitative estimate of the inactivation time course of I Na(T) was also yielded to 0.97 µM (in the Supplementary Information), a value that was noted to be similar to the IC 50 value required for its suppression of I Na(L) , but not for I Na(T) . Pertinent to this reaction scheme is thus that the open-blocked Na V channels tend to be not closed unless the KB-R7943 molecule dissociates from the binding site(s). Meanwhile, the time-dependent block caused by this compound suggests that it preferentially binds to and blocks the open/inactivated state (conformation) of the Na V channels, thereby leading to a destabilization in open conformation [16]. Moreover, although the steady-state I-V relationship of I Na(T) was unaffected during the presence of KB-R7943, this compound diminished the strength of I Na(W) or I Na(R) evoked by respective ascending or descending V ramp . Therefore, whatever ionic mechanisms are involved, the effectiveness of this compound in the perturbations of I Na described herein appear to be independent of an interaction with the NCX exchanging current; hence, it could be viewed to be an additional yet important factor for influencing functional activities of endocrine, neuroendocrine, or neuronal cells (e.g., membrane excitability).
Previous reports have demonstrated that with the increasing duration of the depolarizing pulse, either the magnitude of the NCX exchanging current in frog atrial cells or the Ca 2+ -activated nonselective cationic current in pituitary cells was progressively increased [1,44]. In contrast, the deactivating I Na(L) presented herein was noted to decrease in an exponential fashion as the duration of depolarizing voltage step was prolonged when cells were exposed to Ca 2+ -free Tyrode solution ( Figure 5). The I Na(L) activated by the envelope-of-tail methods showed a decay in a time-dependent manner. Moreover, as the measuring electrode was filled with an internal solution containing a high concentration of EGTA (10 mM), the ability of KB-R7943 to suppress I Na(T) and I Na(L) still remained effective. It is therefore reasonable to assume that the reduction in I Na(L) produced by KB-R7943 is unlikely to be associated with the suppression of NCX exchanging activity, although the NCX exchanging process was earlier demonstrated to be functionally expressed in endocrine or pituitary cells [4,39,[41][42][43]. KB-R7943 has been reported to suppress the reverse mode of NCX1 with effective IC 50 of 1.2-2.4 µM [7]. Moreover, in cultured hippocampal neurons, KB-R7943 exhibits neuroprotection from glutamate-induced excitotoxicity by blocking NMDA receptor-mediated activity (IC 50 = 13.4 µM) as well as by inhibiting complex I in the mitochondrial respiratory chain (IC 50 = 11.4 µM) [8]. Under this scenario, the inhibitory effect of this compound on the magnitude and gating of I Na could be of mechanistic, pharmacological, or even clinical relevance [15,16,70].
It also needs to be mentioned that either instantaneous I Na(W) or I Na(R) evoked by the rising or downsloping (or repolarizing) V ramp , respectively, can be susceptible to being suppressed by adding this compound. Although the overall steady-state I-V relationship of I Na(T) remained unaffected in the presence of KB-R7943, the addition of this compound also had the propensity to suppress the I Na(W) 's strength as well as the I Na(R) magnitude activated by V ramp . Continued exposure to KB-R7943, but still in the presence of Tef or DLT, could reverse the stimulation of I Na(W) ; moreover, either the subsequent addition of Ran or Tef diminished or attenuated, respectively, the KB-R7943-mediated decrease in I Na(R) . Previous reports have shown that the extent of I Na(W) 's strength is linked to the magnitude of background or residual steady Na + currents in many excitable cells [47,48,50,71]. Meanwhile, I Na(R) 's magnitude has also been demonstrated to be associated with high-frequency or varying burst firing of action potentials in excitable cells (e.g., cerebellar Purkinje neurons) [51][52][53][54][55][56]72]. Moreover, the I Na(P) magnitude induced by sinusoidal voltage waveform was also augmented by adding Tef; of note, the further addition of KB-R7943 could attenuate Tef-augmented I Na(P) . The docking results shown herein predicted an interaction of the hNa V 1.2 or hNa v 1.7 channel and the KB-R7943 molecule. This study also showed the ability of benzamil or amiloride to suppress I Na(L) magnitude. As such, the modifications by KB-R7943 of I Na(W) , I Na(R) , and I Na(P) demonstrated herein are important and should not be underestimated. Caution thus needs to be exercised in attributing the action of KB-R7943 or other relevant compounds on the functional activities of excitable cells solely to their suppression in the activity of the NCX exchanging process [6,7,40,46,61,66,67,69,73].  Tainan, Taiwan). Nimodipine, tefluthrin (Tef), tetraethylammonium chloride (TEA), and tetrodotoxin (TTX) were acquired from Sigma (Merck, Taipei, Taiwan); benzamil and amiloride from Tocris (Union Biomed Inc., Taipei, Taiwan); and deltamethrin (DLT, deltamethrin) from MedChemExpress (Asia Bioscience, Taipei, Taiwan). For long-term storage, the stock solution of KB-R7943 was stored at −20 • C in the dark. Culture media (e.g., Ham's F-12 medium), fetal calf serum, horse serum, L-glutamine, and trypsin/EDTA were supplied by HyClone TM (Genchain, Kaohsiung, Taiwan). Other reagents or chemicals were of the highest purity available from commercial sources. Freshly prepared ultrapure water provided by APS Water Services Inc. (Van Nuys, CA, USA) was used in all experiments.

Materials and Methods
The ionic compositions of the external solution (i.e., HEPES-buffered normal Tyrode solution) were as follows (in mM): NaCl 136.5, CaCl 2 1.8, KCl 5.4, MgCl 2 0.53, glucose 5.5, and HEPES 5.5 (pH 7.4 adjusted by adding NaOH). To measure ionic currents flowing through K + currents, the patch electrode was filled with the internal solution comprising (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 (pH adjusted with KOH). To record variable types of voltage-gated Na + current (I Na ), K + ions inside the pipette solution were replaced with equimolar Cs + ions, and the pH was titrated to 7.2 with CsOH. In some experiments designed to highly buffer intracellular Ca 2+ , the internal pipette solution contained EGTA at a concentration of 10 mM. All solutions were prepared by using deionized water which was produced by a Milli-Q ® water purification system (Shih Jhih Technology, Tainan, Taiwan).

Electrophysiological Measurements
Shortly before the experiments, cells were carefully detached from culture dishes, and an aliquot of cell suspension was transferred to a homemade chamber and allowed to settle to the bottom. The chamber was firmly positioned on the stage of a CKX-41 inverted microscope (Olympus; Yuan-Li, Kaohsiung, Taiwan), and cells were immersed at room temperature (20-25 • C) in normal Tyrode solution, the ionic compositions of which are described above. Patch clamp recordings in the whole-cell configuration were performed with the help of either an RK-400 (Bio-Logic, Claix, France) or an Axopatch-200B patch amplifier (Molecular Devices; Bestogen Biotech, New Taipei City, Taiwan) [10,22,74]. Patch electrodes with tip resistances of 3-5 MΩ were made of Kimax ® -51 glass capillaries (#34500-99; Kimble ® ; Dogger, New Taipei City, Taiwan) by using a PP-830 vertical puller (Narishige; Major Instruments, New Taipei City, Taiwan), and then fire-polished with an MF-83 microforge (Narishige). The junction potential, which occurred due to different compositions between extracellular and intracellular solutions, was zeroed shortly before GΩ-seal formation, and the whole-cell data were then corrected.

Whole-Cell Data Recordings with Patch-Clamp Technique
The signal output (i.e., potential and current traces) was monitored on an HM-507 oscilloscope (Hameg, East Meadown, NY, USA) and digitally stored online in an Acer SPIN-5 laptop computer (Yuan-Dai, Tainan, Taiwan) at 10 kHz or more through the Digidata ® 1440-A interface (Molecular Devices, Sunnyvale, CA, USA). During the measurements, the latter device was controlled by pCLAMP TM 10.6 software (Molecular Devices) run under Microsoft ® Windows TM 7 (Redmond, WA, USA). We low-pass filtered current signals at 2 kHz with an FL-4 four-pole Bessel filter (Dagan, Tainan, Taiwan) before they were digitized. Through digital-to-analog conversion, manifold pCLAMP-generated voltage protocols (i.e., rectangular, ramp, or sinusoidal waveforms) were tailored to determine the nonlinear properties of either window Na + current (I Na(W) ), resurgent Na + current (I Na(R) ), or persistent Na + current (I Na(P) ). After the signals were digitally stored, we analyzed them offline by using manifold analytical tools that include LabChart TM 7.0 program (AD Instrument, KYS Technology, Tainan, Taiwan), OriginPro ® 2022b (Microcal; Scientific Formosa, Kaohsiung, Taiwan), and custom macro procedures built under Microsoft ® Excel ® 2021 (Redmond, WA, USA).

Data Analyses
To assess sigmoidal dose-dependent inhibition of KB-R7943 on the peak or transient I Na (I Na(T) ) and sustained or late I Na (I Na(L) ) of the voltage-gated Na + current (I Na ), we placed GH 3 cells in Ca 2+ -free Tyrode solution, and the measuring electrode used presently was filled up with an internal solution containing Cs + . To measure I Na(T) and I Na(L) , we voltage-clamped each examined cell at −100 mV, and the depolarizing voltage command steps to −10 mV for a duration of 30 ms were delivered at a rate of 0.1 Hz. The I Na(T) or I Na(L) amplitudes taken at the start or end-pulse of each depolarizing pulse were respectively measured during the control period (i.e., KB-R7943 was not present) and with cell exposure to different concentrations of KB-R7943 (0.3-30 µM). The IC 50 value required for KB-R7943mediated inhibition of I Na(T) or I Na(L) observed in GH 3 cells was thereafter optimally determined by using a modified Hill function, as follows: In this equation, IC 50 is the concentration required for 50% inhibition, n H is the Hill coefficient, and [KB-R7943] is the KB-R7943 concentration applied. Maximal inhibition (i.e., 1−a) was approximated from this equation. This formula could converge in an appropriate way to give the best-fitting sigmoidal line and the reliable parameter estimates (e.g., IC 50 ).

Curve-Fitting Approximations and Statistical Analyses
Linear or nonlinear curve fitting (e.g., sigmoidal or exponential curve) to each data set was approximated with the least-squares minimization procedure by using manifold maneuvers, which include the "Solver" function embedded in Excel ® 2016 (Microsoft ® ), OriginPro ® 2022b program (OriginLab ® ; Scientific Formosa, Kaohsiung, Taiwan) and MATLAB ® 7.0 (I-Planet Information, Tainan, Taiwan). The averaged results are presented as the mean ± standard error of the mean (SEM) with independent sampling sizes (n) indicating cell numbers from which the observations were properly collected. For two different groups, we used the paired or unpaired Student's t-test. To evaluate the differences among more than two groups (e.g., intertreatment differences), we utilized one-way analysis of variance (ANOVA-1 or ANOVA-2) followed by the post hoc Fisher's least-significance difference method. Statistical analyses were made using the SPSS 20 software package (Asi-aAnalytics, Taipei, Taiwan). Statistical significance was determined at a p-value of < 0.05 ( * , ** , or + are indicated in the figures).