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

Abstract

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 (I_Na), 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 (I_Na(T)) or late component (I_Na(L)) of I_Na with an IC₅₀ value of 11 or 0.9 μM, respectively. Likewise, the dissociation constant for the KB-R7943-mediated block of I_Na on the basis of a minimum reaction scheme was estimated to be 0.97 μM. The presence of benzamil or amiloride could suppress the I_Na(L) magnitude. The instantaneous window Na⁺ current (I_Na(W)) activated by abrupt ascending ramp voltage (V_ramp) was suppressed by adding KB-R7943; however, subsequent addition of deltamethrin or tefluthrin (Tef) effectively reversed KB-R7943-inhibted I_Na(W). With prolonged duration of depolarizing pulses, the I_Na(L) amplitude became exponentially decreased; moreover, KB-R7943 diminished I_Na(L) magnitude. The resurgent Na⁺ current (I_Na(R)) evoked by a repolarizing V_ramp was also suppressed by adding this compound; moreover, subsequent addition of ranolazine or Tef further diminished or reversed, respectively, its reduction in I_Na(R) magnitude. The persistent Na⁺ current (I_Na(P)) activated by sinusoidal voltage waveform became enhanced by Tef; however, subsequent application of KB-R7943 counteracted Tef-stimulated I_Na(P). The docking prediction reflected that there seem to be molecular interactions of this molecule with the hNa_V1.2 or hNa_V1.7 channels. Collectively, this study highlights evidence showing that KB-R7943 has the propensity to perturb the magnitude and gating kinetics of I_Na (e.g., I_Na(T), I_Na(L), I_Na(W), I_Na(R), and I_Na(P)) and that the Na_V channels appear to be important targets for the in vivo actions of KB-R7943 or other relevant compounds.

Keywords: KB-R7943; Na+-Ca2+ exchange; current kinetics; late Na+ current; persistent Na+ current; resurgent Na+ current; transient Na+ current; voltage-gated Na+ current; window Na+ current.

Figure 1

Inhibitory effect of KB−R7943 on voltage-gated Na⁺ current (I_Na) identified from pituitary GH₃ cells. The whole−cell current recordings were conducted in cells bathed in Ca²⁺−free Tyrode solution containing 10 mM tetraethylammonium chloride (TEA) and 0.5 mM CdCl₂, 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 GH₃ cells. The smooth blue or red line drawn represents the goodness-of-fit to the modified Hill equation, as elaborated in Section 4. The IC₅₀ 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).

Figure 2

Effect of benzamil, amiloride, benzamil plus tefluthrin (Tef), and benzamil plus deltamethrin (DLT) on I_Na measured from GH₃ 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).

Figure 3

Effect of KB−R7943 on I_Na(T) evoked by different levels of depolarizing voltage commands measured from GH₃ cells. Average current versus voltage (I−V) relationships of peak amplitude of I_Na(T) under control (filled black squares) and during the exposure to 3 μM KB-R7943 (open red squares). Each cell was depolarized from −80 mV to various potentials ranging from −80 to +40 mV in 10 mV increments for a duration of 30 ms. The I_Na(T) amplitude was measured at the beginning of each voltage pulse. Each point represents the mean ± SEM (n = 7).

Figure 4

Inhibitory effect of KB-R7943 on nonlinear window I_Na (I_Na(W)) activated by abrupt ascend−ng ramp voltage (V_ramp) in GH₃ cells. This set of whole−cell current recordings was undertaken with the tested cell voltage-clamped at −80 mV, and we then applied V_ramp from −100 to +50 mV for a duration of 150 ms on the cell. (A) Exemplar current traces were acquired during the control period (a, black color) and in the presence of 3 μM KB-R7943 (b, red color, KB) or 10 μM KB−R7943 (c, purple color, KB). The voltage clamp protocol applied is shown in the inset, whereas a downward deflection is indicated as the appearance of transient inward current (i.e., instantaneous I_Na(W)) in response to short ascending V_ramp. (B) Summary bar graph showing the effect of KB−R7943 (KB, 3 or 10 μM), KB-R7943 plus deltamethrin (DLT), and KB−R7943 plus tefluthrin (Tef) on the ∆area 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).

Figure 5

Effect of KB−R7943 on both recovery of I_Na(L) (A) and I_Na(L) magnitude evoked by the envelope−of−tail test (B) seen in GH₃ cells. (A) Exemplar current traces taken during the control period (a, absence of KB-R7943) and with presence of 3 μM KB−R7943 (b). Different colors represent the specific current trace evoked by the voltage pulse with increasing duration at the level of −10 mV. The panels shown on the right side denote the expanded records from purple dashed boxes with solid curve arrows on the left side for better illustrations. (B) Relationship of the duration of depolarizing pulse versus the I_Na(L) amplitude at −50 mV acquired in the absence (filled black squares) and presence (open red circles) of 3 μM KB−R7943 (mean ± SEM; n = 7 for each point). Current amplitude was measured at the end of voltage pulse at the level of −50 mV, and the absolute value of current amplitude is illustrated. (C) KB−R7943−mediated changes in I_Na(L) amplitude evoked by the envelope−of−tail test (mean ± SEM; n = 7 for each point). The relationships of the pulse duration versus the relative amplitude of I_Na(L) are illustrated with or without cell exposure to 3 μM KB−R7943. The relative amplitude appearing at the y−axis was measured in situations where I_Na(L) amplitude at the end of depolarizing pulse from −100 to −10 mV was divided by the tail current at the end of the voltage step taken following a return to −50 mV. The decaying rate of I_Na(L) evoked during the envelope−of−tail occurred in a single exponential function.

Figure 6

Inhibitory effect of KB−7943 on instantaneous resurgent Na⁺ current (I_Na(R)) identified from GH₃ cells. The experiments used to evoke non-linear I_Na(R) were designed to consist of a conditioning pulse step from −80 to +30 mV with a duration of 30 ms followed by a 1 s descending V_ramp from +30 to −80 mV (i.e., ramp pulse of −0.11 mV/ms). (A) Exemplar relationship of the current amplitude versus the membrane potential taken in the control period (a, black color) and during exposure to 3 μM KB−7943 (b, red color). Inset shows the voltage clamp protocol applied. (B) Summary graph disclosing effects of KB−7943 (KB, 1 or 3 μM), KB−7943 plus ranolazine (Ran), and KB−7943 plus tefluthrin (Tef) on the amplitude of I_Na(R) in response to the descending (repolarizing) V_ramp (mean ± SEM; n = 7 for each yellow bar). Each current amplitude evoked by the downsloping V_ramp was taken at the level of −5 mV. * Significantly different from control (p < 0.05), ** significantly different from KB-7943 (1 μM) alone group (p < 0.05), and ⁺ significantly different from KB−7943 (3 μM) alone group (p < 0.05).

Figure 7

Attenuating effect of KB−R7943 on tefluthrin-stimulated I_Na(P) activated in response to sinusoidal voltage waveform. The tested cell was held at −80 mV, and the voltage clamp protocol designed to consist of sinusoidal voltages between −100 and 0 mV with a rate of 5 Hz for a duration of 1 sec was afterwards applied to it. (A) Exemplar current traces obtained in the control period (a, black color, i.e., neither Tef nor KB (KB−7943) was present), and during cell exposure to either 10 μM Tef (b, orange color) or 10 μM Tef plus 3 μM KB−R7943 (c, purple color). The asterisk in each panel indicates a transient inward deflection corresponding with the occurrence of I_Na(P) activated in response to sinusoidal voltage waveform. (B) Effect of tefluthrin (Tef) and Tef plus KB−R7943 (KB, 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).

Figure 8

Molecular docking of the hNa_V1.7 channel and the KB-R7943 molecule. The chemical structure of KB-R7943 was acquired from PubChem (compound CID: 9823846), whereas the protein structure of hNa_V1.7 was from RCB PDB (ID: 5EK0). We docked the KB-R7943 molecule to hNa_V1.7 with the help of PyRx software (http://pyrx.sourceforge.io/) (accessed on 16 September 2022), and the diagram of the interaction between the hNa_V1.7 channel and the KB-R7943 molecule was then generated from LigPlot⁺ (http://www.ebi.ac.uk/thornton-srv/software/LIGPLOT/) (accessed on 16 September 2022). Note that the red arcs on which red small bars are faced and radiated toward the ligand (i.e., KB-R7943 molecule) represent the hydrophobic interactions, while green dotted lines residing in amino acid residue (i.e., Ser1681(B), Ser1681(C), Thr1709(C) and Met1877(C)) show the formation of hydrogen bonds.

Figure 9

Molecular docking of the hNa_V1.2 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_V1.2 was from RCB PDB (ID: 2KAV). We docked the KB-R7943 molecule to hNa_V1.2 with PyRx, and diagram of the interaction between the hNa_V1.2 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.