The Effectiveness of Isoplumbagin and Plumbagin in Regulating Amplitude, Gating Kinetics, and Voltage-Dependent Hysteresis of erg-mediated K+ Currents

Isoplumbagin (isoPLB, 5-hydroxy-3-methyl-1,4-naphthoquinone), a naturally occurring quinone, has been observed to exercise anti-inflammatory, antimicrobial, and antineoplastic activities. Notably, whether and how isoPLB, plumbagin (PLB), or other related compounds impact transmembrane ionic currents is not entirely clear. In this study, during GH3-cell exposure to isoPLB, the peak and sustained components of an erg (ether-à-go-go related gene)-mediated K+ current (IK(erg)) evoked with long-lasting-step hyperpolarization were concentration-dependently decreased, with a concomitant increase in the decaying time constant of the deactivating current. The presence of isoPLB led to a differential reduction in the peak and sustained components of deactivating IK(erg) with effective IC50 values of 18.3 and 2.4 μM, respectively, while the KD value according to the minimum binding scheme was estimated to be 2.58 μM. Inhibition by isoPLB of IK(erg) was not reversed by diazoxide; however, further addition of isoPLB, during the continued exposure to 4,4′-dithiopyridine, did not suppress IK(erg) further. The recovery of IK(erg) by a two-step voltage pulse with a geometric progression was slowed in the presence of isoPLB, and the decaying rate of IK(erg) activated by the envelope-of-tail method was increased in its presence. The strength of the IK(erg) hysteresis in response to an inverted isosceles-triangular ramp pulse was diminished by adding isoPLB. A mild inhibition of the delayed-rectifier K+ current (IK(DR)) produced by the presence of isoPLB was seen in GH3 cells, while minimal changes in the magnitude of the voltage-gated Na+ current were demonstrated in its presence. Moreover, the IK(erg) identified in MA-10 Leydig tumor cells was blocked by adding isoPLB. Therefore, the effects of isoPLB or PLB on ionic currents (e.g., IK(erg) and IK(DR)) demonstrated herein would be upstream of our previously reported perturbations on mitochondrial morphogenesis or respiration. Taken together, the perturbations of ionic currents by isoPLB or PLB demonstrated herein are likely to contribute to the underlying mechanism through which they, or other structurally similar compounds, result in adjustments in the functional activities of different neoplastic cells (e.g., GH3 and MA-10 cells), presuming that similar in vivo observations occur.


Introduction
Isoplumbagin (isoPLB, 5-hydroxy-3-methyl-1,4-naphthoquinone) is a naturally occurring quinone from Lawsonia inermis [1] or Plumbago europarea [2]. Similar to isoPLB, plumbagin (PLB, 5-hydroxy-2-methyl-1,4-naphthoquinone), another hydroxyl-1,4-napththoquinone, is an alkaloid obtained from the roots of the plants of the Plumbago genus. IsoPLB and PLB have recently been demonstrated to exert anti-neoplastic activity against an array of cancers, including oral or tongue squamous cell carcinoma, glioblastoma, non-small cell lung carcinoma, and breast, cervical, endometrial, pancreatic, and prostate cancers . Nevertheless, PLB was shown to induce apoptotic changes in lung cancer or neuronal cells via either caspase-9 activation or the enhancement of mitochondrial-associated ROS [10,15,25,26]. PLB has been reported to modify the release of pituitary gonadotropin [27]. Previous studies have shown the ability of 2-mercaptophenyl-1,4-naphthoquinone, a naphthoquinone derivative, to overcome the elevation of intracellular Ca 2+ in platelets caused by ADP and collagen [28], as well as to suppress the amplitude of Ca 2+ -activated and voltage-gated K + currents in pituitary tumor (GH 3 ) cells [29]. However, to our knowledge, detailed information is limited regarding the possible perturbations of isoPLB and its related compounds on different types of ionic currents, despite growing awareness of their ability to exert anti-neoplastic actions.
The erg (ether-à-go-go related gene)-mediated K + current (I K(erg) ), the necessary components for which are encoded by three different subfamilies of the KCNH gene, is able to generate the pore-forming α-subunit of erg-mediated K + (i.e., K erg or K V 11) channels [30,31]. This type of K + current is also thought to constitute a cloned counterpart of the rapidly activating delayed-rectifying K + current residing in cardiac myocytes, where the KCNH2 gene encodes the pore-forming α-subunit of the K V 11.1 channels, commonly identified as hERG [31][32][33]. The I K(erg) current is intrinsically present in neurons or varying types of excitable cells, such as neuroendocrine or endocrine cells. Of note, it can be engaged in the maintenance of resting potential and in modifications of the subthreshold excitability [34][35][36][37][38][39][40][41][42][43][44][45][46]. Previous work has also shown the effectiveness of the I K(erg) magnitude in regulating the apoptosis and proliferation of many types of neoplastic cells [47][48][49][50][51][52][53][54]. However, whether or how the presence of isoPLB and PLB changes the magnitude of this type of K + current is largely unclear.
Earlier reports have demonstrated that pituitary cells (e.g., GH 3 lactotrophs), in addition to possessing voltage-gated K + currents, can functionally express I K(erg) , the magnitude of which can perturb the functional activities of these cells [30,35,36,[55][56][57][58][59][60][61][62]. This type of ionic current is unique, and is characterized by voltage-dependent activation and deactivation with an inwardly rectifying property. The magnitude of the current has been proposed to be a main determinant of the resting potential and, therefore, of membrane excitability [34,36,60]. Any modifications of this K erg -channel activity also have the propensity to alter the coupling of stimulus and secretion in these cells [30,36,55,60,63].
In view of the initiatives elaborated above, the objective of this work is to explore whether isoPLB, PLB, or other related compounds are capable of interacting with plasmalemmal ion channels to perturb the amplitude, gating, and voltage-dependent hysteresis (V hys ) of membrane ionic currents, particularly the erg-mediated K + current (I K(erg) ). Our results demonstrate that the presence of isoPLB inhibits hyperpolarization-evoked I K(erg) in a concentration-, time-, state-, and hysteresis-dependent manner in pituitary GH 3 cells. The I K(erg) intrinsically present in MA-10 Leydig tumor cells is also sensitive to inhibition by isoPLB.

Patch-Clamp Recordings: Electrophysiological Measurements
Shortly before each experiment, we gently dispersed GH 3 or MA-10 cells with a 1% trypsin-EDTA solution, and an aliquot of suspension containing clumps of cells was transferred to a home-made chamber affixed to the working stage of a DM-IL inverted microscope (Leica; Major Instruments, Kaohsiung, Taiwan). We bathed cells at room temperature (20-25 • C) in HEPES-buffered normal Tyrode's solution containing 1.8 mM CaCl 2 , the composition of which is described above. The pipettes that we used were prepared from Kimax ® -51 borosilicate capillaries (#34500; Dogger, Tainan, Taiwan) by using either a vertical PP-83 puller (Narishige; Taiwan Instrument, Tainan, Taiwan) or a horizontal P-97 puller (Sutter, Novato, CA, USA), and their tips were fire-polished with an MF-83 microforge (Narishige). For efficient recordings, we chose electrodes with tip resistances of 3-5 MΩ, as they were filled with different internal solutions as stated above. Macroscopic ionic currents passing through the whole cell (i.e., whole-cell mode) with a modified patch-clamp technique were measured using either an RK-400 (Bio-Logic, Claix, France) or an Axopatch TM -200B (Molecular Devices ® ; Bestogen, New Taipei City, Taiwan) patch amplifier [57,66,68]. All potentials were corrected for liquid junction potentials that would arise at the pipette tip when the composition of the internal solution differed from that in the bath. The compounds tested were either applied through perfusion or added to the bath in order to achieve the final concentration indicated. In order to record the I K(erg) , we voltage-clamped the examined cell at a holding potential of −10 mV before a long-lasting hyperpolarizing step was delivered.

Data Recordings
Data acquisition was performed using either pCLAMP 10.6 (Molecular Devices), LabChart 7.1 (PowerLab; ADInstruments, Kuoyang, New Taipei City, Taiwan), or Hantek 6022BL (Qingdao, China). Current signals were low-pass filtered at 1 or 3 kHz. Analog signals were monitored and stored online at 5-10 kHz via analog-to-digital (AD) conversion in an ASUSPRO-BU401LG laptop computer (ASUS, Tainan, Taiwan), which was equipped with a low-noise Digidata ® 1440A device (Molecular Devices). With digital-to-analog (DA) conversion, the various voltage waveforms were specifically designed to determine the steady-state current-voltage (I-V) relationship, the recovery of the current block with a twostep voltage protocol in a geometric progression, or the V hys of the ionic currents specified (e.g., I K(erg) ). The data digitally acquired during the experiments were subsequently analyzed by different analytical tools, such as OriginPro ® 2021 (OriginLab; Scientific Formosa, Kaohsiung, Taiwan), LabChart 7.1 (ADInstruments), and custom-built macros in Excel ® 2021 spreadsheets under Windows 10 (Microsoft, Redmond, WA, USA).

Whole-Cell Data Analyses
To assess the percentage inhibition of isoPLB on the I K(erg) amplitude, each tested cell was 1-s hyperpolarized from −10 to −90 mV, and the current magnitudes were measured at the beginning (i.e., peak amplitude) or end-pulse (i.e., sustained amplitude) of the voltage pulse during cell exposure to different isoPLB concentrations and then compared. The concentration of isoPLB required to suppress 50% of the peak or sustained component in the current amplitude ( Figure 1C) was determined using a Hill function: where y = percentage inhibition (%); [C] = the concentration of isoPLB used; n H = the Hill coefficient; IC 50 = half-maximal inhibition; E max = isoPLB-induced maximal inhibition of I K(erg) (peak or sustained component). The inhibitory effect of isoPLB on I K(erg) seen in GH 3 cells apparently occurred in a time-dependent manner and it can reasonably be explained by a state-dependent blocker which can preferentially bind to the open state (or conformation) of the K erg channel. As such, from a simplified assumption, the first-order reaction scheme in which the isoPLB molecule interacts with the K erg channel is given as follows: where [isoPLB] indicates the isoPLB concentration given, α or β is the voltage-dependent rate constant needed for the opening and closing of the K erg channels, respectively, and, k +1 * or k −1 respectively represents the forward (on or bound) or backward (off or un-bound) rate constants of isoPLB binding, respectively. It means that the value of k +1 *·[isoPLB] changes as a function of the isoPLB concentration, while k −1 is independent of the isoPLB concentration per second. The forward and reverse rate constants (i.e., k +1 * and k −1 ) were determined from the time constants of the current decay changed upon step hyperpolarization from −10 to −90 mV with a duration of 1 s. The deactivation time constant (τ deact ) of the resulting current was satisfactorily estimated by fitting the decaying trajectory of each current trace with a single exponential. The evolving rate constants (1/τ deact ) would be then evaluated using the following equation: In this linear relationship, k +1 * or k −1 were respectively obtained either from the slope (i.e., ∆(1/∆τ)/∆([isoPLB]) or from the y-axis intercept (i.e., y intercept) at [isoPLB] = 0 of the linear regression into which the relationship of the 1/∆τ value versus the isoPLB concentrations was interpolated. ∆τ indicates the difference in the τ deact value obtained in situations where the τ deact values during exposure to different isoPLB concentrations (1-50 µM) were subtracted from those in the presence of 100 µM isoPLB ( Figure 1B). A measure of the dissociation constant (K D ), which is equal to k −1 divided by k +1 * can thereby be calculated. The forward and reverse rate constants (i.e., k+1 * and k − 1) were determined from the time constants of the current decay changed upon step hyperpolarization from −10 to −90 mV with a duration of 1 sec. The deactivation time constant (τdeact) of the resulting current was satisfactorily estimated by fitting the decaying trajectory of each current trace with a single exponential. The evolving rate constants (1/τdeact) would be then evaluated using the following equation: In this linear relationship, k+1 * or k − 1 were respectively obtained either from the slope (i.e., ∆(1/∆τ)/∆([isoPLB]) or from the y-axis intercept (i.e., y intercept) at [isoPLB] = 0 of the linear regression into which the relationship of the 1/∆τ value versus the isoPLB concentrations was interpolated. ∆τ indicates the difference in the τdeact value obtained in situations where the τdeact values during exposure to different isoPLB concentrations (1-50 μM) were subtracted from those in the presence of 100 μM isoPLB ( Figure 1B). A measure of the dissociation constant (KD), which is equal to k − 1 divided by k+1 * can thereby be calculated.

Curve Fitting Approximations and Statistical Analyses
The curve-fitting (linear or nonlinear) to experimental data acquired in this work was conducted with the goodness-of-fit using different methods, such as pCLAMP 10.7 (Molecular Devices), OriginPro 2021 (OriginLab), and Microsoft Excel ® 2021 (i.e., "Solver" add-in) under Microsoft Office 365 (Redmond, WA, USA). The whole-cell data are presented as mean ± standard error of the mean (SEM) with the size of experimental observations (n) indicative of cell numbers from which the results were taken, and error bars are plotted as SEM. The data distribution was found to satisfy the tests for normality. The Student's t-test (paired or unpaired) between two groups was first used for the statistical analyses. When the differences among different groups needed to be testified, we further conducted an analysis of variance (ANOVA-1 or ANOVA-2), which was then followed by

Curve Fitting Approximations and Statistical Analyses
The curve-fitting (linear or nonlinear) to experimental data acquired in this work was conducted with the goodness-of-fit using different methods, such as pCLAMP 10.7 (Molecular Devices), OriginPro 2021 (OriginLab), and Microsoft Excel ® 2021 (i.e., "Solver" add-in) under Microsoft Office 365 (Redmond, WA, USA). The whole-cell data are presented as mean ± standard error of the mean (SEM) with the size of experimental observations (n) indicative of cell numbers from which the results were taken, and error bars are plotted as SEM. The data distribution was found to satisfy the tests for normality. The Student's t-test (paired or unpaired) between two groups was first used for the statistical analyses. When the differences among different groups needed to be testified, we further conducted an analysis of variance (ANOVA-1 or ANOVA-2), which was then followed by a post-hoc Fisher's least-significance different test for multiple-range comparisons. Probability with p < 0.05 was considered to indicate statistical difference, unless noted otherwise.

Effect of IsoPLB on I K(erg) Recorded from GH 3 Cells
In the initial stage of the measurements, we used the whole-cell configuration of the patch-clamp technique to evaluate if the presence of isoPLB leads to any perturbations on ionic currents (e.g., I K(erg) ) residing in GH 3 cells. In order to measure the current flowing through I K(erg) , we placed the cells to be bathed in a high-K + , Ca 2+ -free external solution which contained 1 µM tetrodotoxin (TTX) and 0.5 mM CdCl 2 , and we then backfilled the recording pipette using a K + -enriched (145 mM) internal solution. TTX or CdCl 2 was used to suppress the magnitude of voltage-gated Na + or Ca 2+ currents [46,69], respectively, in order to avoid any interference with measurements of I K(erg) . As the whole-cell mode was established, we voltage-clamped the tested cell at the potential of −10 mV in voltage clamp mode and the hyperpolarizing step was thereafter applied (1 s in duration) to −90 mV for the elicitation of deactivating I K(erg) with a slowly decaying time course, as demonstrated previously [46,[57][58][59]67].
As illustrated in Figure 1A, one minute after cells were exposed to isoPLB at a concentration of 1 or 3 µM, the amplitude of I K(erg) evoked by such long-lasting hyperpolarizing step progressively became decreased, and the decaying rate of the evolving current concurrently was reduced. For example, cell exposure to 3 µM isoPLB significantly decreased the peak and sustained components of I K(erg) to 99 ± 11 pA (n = 8, p < 0.05) and 48 ± 8 pA (n = 8, p < 0.05) from the control values of 147 ± 22 pA and 96 ± 11 pA (n = 8), respectively. Meanwhile, under exposure to 3 µM isoPLB, the value for the deactivation time constant (τ deact ) of I K(erg) was also lengthened from 101 ± 18 to 287 ± 25 ms (n = 8, p < 0.05). After the washout of isoPLB, the peak and sustained amplitudes of I K(erg) evoked by membrane hyperpolarization were returned to 142 ± 21 pA (n = 7) and 92 ± 9 pA (n = 7), respectively. Likewise, similar results were obtained during cell exposure to 3 µM PLB in seven different examined cells.
It was noted that the observed effect of isoPLB on I K(erg) could not only emerge instantaneously, but could also emerge in a time-and concentration-dependent fashion. A linear relationship between the 1/∆τ deact value and the isoPLB concentration is therefore illustrated in Figure 1B. The forward (k +1 *) or backward (k −1 ) rate constant derived from the minimum binding and unbinding scheme were thereafter estimated to be 0.39 s −1 µM −1 and 1.01 s −1 , respectively; consequently, the value of the dissociation constant (K D = k −1 /k +1 *) was optimally calculated to be 2.58 µM. This value was noticed to be similar to the IC 50 value (indicated in Figure 1C) required for an isoPLB-induced block of sustained I K(erg) measured at the end of the hyperpolarizing step. Figure 1C illustrates that cell exposure to isoPLB in the bath can concentrationdependently depress the amplitude of the peak or sustained I K(erg) evoked in response to the long-lasting hyperpolarizing step. According to the modified Hill equations detailed under Materials and Methods, the IC 50 values needed for the isoPLB-mediated inhibitory effects on peak and sustained I K(erg) were estimated to be 18.3 or 2.4 µM, respectively. The results reflect that isoPLB exercises a depressant action on the peak and sustained I K(erg) present in GH 3 cells and that this compound tends to be selective for sustained over peak I K(erg) evoked by the hyperpolarizing step.

Steady-State I-V Relationship of Peak and Sustained I K(erg) Caused by IsoPLB
We continued to examine if isoPLB-mediated changes in I K(erg) amplitude can occur at different levels of command voltage pulse. In these experiments, we held the tested cell at −10 mV and a set of voltage steps ranging between −100 and 0 mV with a duration of 1 s was imposed. Figure 2 illustrates the average I-V relationship of the peak (black symbols) and sustained (red symbols) components of I K(erg) with or without cell exposure to 3 µM isoPLB. Likewise, one minute after cell exposure to 3 µM isoPLB, the whole-cell conductance of peak I K(erg) measured between −100 and −60 mV was significantly reduced to 2.12 ± 0.08 nS (n = 8, p < 0.05) from a control value of 2.81 ± 0.09 nS (n = 8). The results indicate that the application of this compound (3 µM) can effectively suppress the peak and sustained components of I K(erg) measured throughout the entire voltage range imposed.
Biomedicines 2022, 10, x FOR PEER REVIEW 7 of 21 symbols) and sustained (red symbols) components of IK(erg) with or without cell exposure to 3 μM isoPLB. Likewise, one minute after cell exposure to 3 μM isoPLB, the whole-cell conductance of peak IK(erg) measured between −100 and −60 mV was significantly reduced to 2.12 ± 0.08 nS (n = 8, p < 0.05) from a control value of 2.81 ± 0.09 nS (n = 8). The results indicate that the application of this compound (3 μM) can effectively suppress the peak and sustained components of IK(erg) measured throughout the entire voltage range imposed.

Comparisons among Effect of IsoPLB, PLB, 4,4′-Dithiodipyridine (DTDP), Chlorotoxin, Azimilide, IsoPLB plus Diazoxide, and DTDP plus IsoPLB on Hyperpolarization-Evoked IK(erg) in GH3 Cells
The effects of isoPLB, PLB, DTDP, chlorotoxin, azimilide, isoPLB plus diazoxide, and DTDP plus isoPLB on the amplitude of IK(erg) in GH3 ells were further assessed and compared. As demonstrated in Figure 3, the application of DTDP (10 μM) or azimilide (10 μM) was able to suppress IK(erg) amplitude, while chlorotoxin, a blocker of Cl − channels [70], was ineffective at inhibiting it. Azimilide is reported to be an inhibitor of IK(erg) [67,71], and DTDP is a lipophilic sulfhydryl oxidizing agent [72,73]. Moreover, we found that with continued exposure to 3 μM isoPLB, the subsequent addition of diazoxide was noticed to have little or no effect on the isoPLB-mediated block of IK(erg) in these cells. Diazoxide was shown to enhance the activity of ATP-sensitive K + (KATP) channels [74,75]. The subsequent application of 3 μM isoPLB, while still in the continued presence of 10 μM DTDP, did not suppress IK(erg) further. These results suggest that isoPLB could share a similar mechanism Figure 2. Inhibitory effect of isoPLB on steady-state I-V relationship of hyperpolarization-activated I K(erg) identified in GH 3 cells. In this series of experiments, we placed cells in a high-K + , Ca 2+ -free solution which contained 1 µM TTX and 0.5 mM CdCl 2 , and the pipette used for recordings was thereafter filled with a K + -enriched solution. We held the examined cell at −10 mV and a set of command potentials ranging between −100 and 0 mV with a duration of 1 s was imposed on it.
(A) Superimposed current traces acquired in the control period (i.e., absence of isoPLB) (upper) and during cell exposure to 3 µM isoPLB (lower). The voltage-clamp protocol is indicated in the top part, and potential traces shown in different colors correspond with currents which were evoked by the same level of step command. In (B,C), average I-V relationships of the peak (black squares) or sustained component (red squares) of I K(erg) attained in the absence (solid symbols) and presence (open symbols) of 3 µM isoPLB were demonstrated, respectively. The peak and sustained amplitudes of I K(erg) were taken at the beginning and end-pulse of each step command applied. Each point represents the mean ± SEM (n = 7-8). The analyses with or without the addition of isoPLB among different levels of potentials given were performed in (B,C) with ANOVA-2 analysis (p < 0.05). The effects of isoPLB, PLB, DTDP, chlorotoxin, azimilide, isoPLB plus diazoxide, and DTDP plus isoPLB on the amplitude of I K(erg) in GH 3 ells were further assessed and compared. As demonstrated in Figure 3, the application of DTDP (10 µM) or azimilide (10 µM) was able to suppress I K(erg) amplitude, while chlorotoxin, a blocker of Cl − channels [70], was ineffective at inhibiting it. Azimilide is reported to be an inhibitor of I K(erg) [67,71], and DTDP is a lipophilic sulfhydryl oxidizing agent [72,73]. Moreover, we found that with continued exposure to 3 µM isoPLB, the subsequent addition of diazoxide was noticed to have little or no effect on the isoPLB-mediated block of I K(erg) in these cells. Diazoxide was shown to enhance the activity of ATP-sensitive K + (K ATP ) channels [74,75]. The subsequent application of 3 µM isoPLB, while still in the continued presence of 10 µM DTDP, did not suppress I K(erg) further. These results suggest that isoPLB could share a similar mechanism of action to DTDP with respect to their block of I K(erg) observed in GH 3 cells, although neither the activity of K ATP nor Cl − channels residing in these cells is predominantly linked to isoPLB-induced block of I K(erg) . of action to DTDP with respect to their block of IK(erg) observed in GH3 cells, although neither the activity of KATP nor Cl − channels residing in these cells is predominantly linked to isoPLB-induced block of IK(erg). In these experiments, we placed cells in a high-K + , Ca 2+ -free solution containing 1 μM TTX and 0.5 mM CdCl2, and the recording electrode was filled up with K + -containing solution. The tested cell was held at −10 mV and a 1-sec step hyperpolarization to −90 mV was subsequently applied to evoke IK(erg). The current amplitude taken during cell exposure to different tested compounds was measured at the beginning of the hyperpolarizing step. Each bar represents the mean ± SEM (n = 7-8). Statistical analyses among different groups were performed with ANOVA-1 (p < 0.05). * Significantly different from control (p < 0.05). isoPLB: isoplumbagin; PLB: plumbagin; and DTDP: 4,4′-dithiodipyridine.

Slowing in Recovery from IK(erg) Block Caused by IsoPLB in GH3 Cells
We continued to explore if the presence of isoPLB could alter the recovery from the block of IK(erg). In this set of experiments, we measured the recovery from the current block by using a standard gapped pulse protocol in which the interpulse interval varies with a geometric progression. The ratio of the amplitude of the second and first peak IK(erg) (i.e., relative amplitude) evoked upon a 1-sec hyperpolarizing pulse to −100 mV from a holding potential of −10 mV was imposed as a measure of the degree of recovery from the IK(erg) block. The results appearing in Figure 4 show that, in the control period (i.e., absence of isoPLB), the peak amplitude of IK(erg) was fully restored from the block when the interpulse interval was set at 1 sec or above. Moreover, the time course of recovery from the current block achieved without and without cell exposure to 3 μM isoPLB was satisfactorily fitted by a single exponential, and time constants of 8.2 ± 2.2 (n = 8) and 22.4 ± 5.1 msec (n = 8) were derived, respectively. As such, the experimental results lead us to reflect that the presence of isoPLB produces an appreciable lengthening in the recovery from the block of peak IK(erg) seen in GH3 cells. , chlorotoxin, azimilide, isoPLB plus diazoxide, and DTDP plus isoPLB on I K(erg) recorded from GH 3 cells . In these experiments, we placed cells in a high-K + , Ca 2+ -free solution containing 1 µM TTX and 0.5 mM CdCl 2 , and the recording electrode was filled up with K + -containing solution. The tested cell was held at −10 mV and a 1-s step hyperpolarization to −90 mV was subsequently applied to evoke I K(erg) . The current amplitude taken during cell exposure to different tested compounds was measured at the beginning of the hyperpolarizing step. Each bar represents the mean ± SEM (n = 7-8). Statistical analyses among different groups were performed with ANOVA-1 (p < 0.05). * Significantly different from control (p < 0.05). isoPLB: isoplumbagin; PLB: plumbagin; and DTDP: 4,4 -dithiodipyridine.

Slowing in Recovery from I K(erg) Block Caused by IsoPLB in GH 3 Cells
We continued to explore if the presence of isoPLB could alter the recovery from the block of I K(erg) . In this set of experiments, we measured the recovery from the current block by using a standard gapped pulse protocol in which the interpulse interval varies with a geometric progression. The ratio of the amplitude of the second and first peak I K(erg) (i.e., relative amplitude) evoked upon a 1-s hyperpolarizing pulse to −100 mV from a holding potential of −10 mV was imposed as a measure of the degree of recovery from the I K(erg) block. The results appearing in Figure 4 show that, in the control period (i.e., absence of isoPLB), the peak amplitude of I K(erg) was fully restored from the block when the interpulse interval was set at 1 s or above. Moreover, the time course of recovery from the current block achieved without and without cell exposure to 3 µM isoPLB was satisfactorily fitted by a single exponential, and time constants of 8.2 ± 2.2 (n = 8) and 22.4 ± 5.1 ms (n = 8) were derived, respectively. As such, the experimental results lead us to reflect that the presence of isoPLB produces an appreciable lengthening in the recovery from the block of peak I K(erg) seen in GH 3 cells.  The relative amplitude achieved in the ordinate was taken when the second amplitude evoked by a 1-sec hyperpolarizing voltage-clamp command from −10 to −100 mV was divided by the first. The smooth curve without and with cell exposure to 3 μM isoPLB was least-squares fitted to a single exponential with time constants of 8.2 and 22.4 msec, respectively. The y axis is illustrated with a logarithmic scale. Of notice, the presence of isoPLB can lead to a prolongation of recovery from the IK(erg) block evoked by such a two-step voltage command protocol.

Modification by IsoPLB of the Time Course of IK(erg) Evoked by the Envelope-of-Tail Test
Earlier studies have demonstrated that there was a time-dependent change in IK(erg) activation during the envelope-of-tail test [76,77]. In this set of envelope-of-tail experiments, we evoked an inward deactivating and an activating IK(erg) elicited by varying durations of hyperpolarizing step (from 4 msec to 8.192 sec with a geometric progression) to . Modification by isoPLB of the recovery of the I K(erg) block seen in GH 3 cells. These measurements were undertaken in cells bathed in a high-K + , Ca 2+ -free solution, and the recording electrode was filled with a K + -containing solution. As the whole-cell configuration was firmly established, we imposed a two-step voltage protocol with a geometric progression onto the examined cells.
(A) Superimposed current traces attained in the control period (i.e., absence of isoPLB) and during cell exposure to 3 µM isoPLB. The uppermost part in (A,B) shows the pulse protocol applied. In (B), the potential and current traces are the expanded records from the dashed box in (A). (C) Relationship of the interpulse interval versus the relative amplitude obtained in the absence (solid blue circles) and presence (solid orange squares) of 3 µM isoPLB (mean ± SEM; n = 8 for each point). The relative amplitude achieved in the ordinate was taken when the second amplitude evoked by a 1-s hyperpolarizing voltage-clamp command from −10 to −100 mV was divided by the first. The smooth curve without and with cell exposure to 3 µM isoPLB was least-squares fitted to a single exponential with time constants of 8.2 and 22.4 msec, respectively. The y axis is illustrated with a logarithmic scale. Of notice, the presence of isoPLB can lead to a prolongation of recovery from the I K(erg) block evoked by such a two-step voltage command protocol.

Modification by IsoPLB of the Time Course of I K(erg) Evoked by the Envelope-of-Tail Test
Earlier studies have demonstrated that there was a time-dependent change in I K(erg) activation during the envelope-of-tail test [76,77]. In this set of envelope-of-tail experiments, we evoked an inward deactivating and an activating I K(erg) elicited by varying durations of hyperpolarizing step (from 4 ms to 8.192 s with a geometric progression) to −90 mV from −10 mV. As can be seen in Figure 5, the relationship of the relative amplitude versus the pulse duration with or without the application of 10 µM isoPLB was established. In keeping with previous observations [76], the envelope-of-tail test tailored for the elicitation of I K(erg) in this study was observed to reveal a time-dependent exponential decay in the ratio of the relative amplitude (i.e., I act /I deac ) evoked during the durations between 4 ms and 8.192 s in a geometric progression, with or without the application of this compound (10 µM).
With GH 3 -cell exposure to 10 µM isoPLB, the time constant of I K(erg) activated by the envelope-of-tail method was significantly decreased to 21.2 ± 1.2 ms (n = 7, p < 0.05) from a control value of 44.3 ± 2.1. It is conceivable from the present observations, therefore, that isoPLB addition can produce an evident shortening in the time course of I K(erg) activation evoked with the envelope-of-tail voltage protocol observed in GH 3 cells.
Biomedicines 2022, 10, x FOR PEER REVIEW 10 of 21 −90 mV from −10 mV. As can be seen in Figure 5, the relationship of the relative amplitude versus the pulse duration with or without the application of 10 μM isoPLB was established. In keeping with previous observations [76], the envelope-of-tail test tailored for the elicitation of IK(erg) in this study was observed to reveal a time-dependent exponential decay in the ratio of the relative amplitude (i.e., Iact/Ideac) evoked during the durations between 4 msec and 8.192 sec in a geometric progression, with or without the application of this compound (10 μM). With GH3-cell exposure to 10 μM isoPLB, the time constant of IK(erg) activated by the envelope-of-tail method was significantly decreased to 21.2 ± 1.2 msec (n = 7, p < 0.05) from a control value of 44.3 ± 2.1. It is conceivable from the present observations, therefore, that isoPLB addition can produce an evident shortening in the time course of IK(erg) activation evoked with the envelope-of-tail voltage protocol observed in GH3 cells. shows the voltage-clamp protocol delivered. (C) Relationship of the relative amplitude versus pulse duration obtained with or without the application of 10 μM isoPLB (mean ± SEM; n = 7 for each point). The relative amplitude appearing at the y axis was measured, when the inwardly decaying current (i.e., Iact) following return to −10 mV was divided by peak IK(erg) (i.e., Ideact) in response to membrane hyperpolarizing pulses with varying pulses in a geometric progression. Of note, the relationship is illustrated with a semi-logarithmic plot. The smooth curve without or with the addition of 10 μM isoPLB indicates the best fit to single exponential function with a time constant of 44.3 or 21.2 msec, respectively. The decaying rate of IK(erg) evoked during the envelope-of-tail occurred in single exponential function, and the presence of isoPLB yielded an increase in the decay rate of IK(erg) evoked upon such envelope-of-tail test.

Modification by IsoPLB of the Vhys Behavior of IK(erg) Activated by Inverted Double Ramp Voltage (Vramp)
Earlier reports have demonstrated the capability of IK(erg)'s Vhys strength to perturb either various patterns of bursting, firing, or action potential configurations intrinsically in different types of excitable cells [31,[36][37][38][39][40][41][42][43][44]46]. Therefore, we next assessed whether and how the exposure of isoPLB could modify the Vhys strength of the IK(erg) evoked with double (i.e., inverted isosceles-triangular) Vramp. In this series of experiments, we voltage-clamped . Of note, the x axis for the potential and current tracings appearing in (A,B) is scaled with a geometric progression. * indicates the I act of hyperpolarization-evoked I K(erg) and ** indicates the I deact of the current. The uppermost part in (A,B) shows the voltage-clamp protocol delivered. (C) Relationship of the relative amplitude versus pulse duration obtained with or without the application of 10 µM isoPLB (mean ± SEM; n = 7 for each point). The relative amplitude appearing at the y axis was measured, when the inwardly decaying current (i.e., I act ) following return to −10 mV was divided by peak I K(erg) (i.e., I deact ) in response to membrane hyperpolarizing pulses with varying pulses in a geometric progression. Of note, the relationship is illustrated with a semi-logarithmic plot. The smooth curve without or with the addition of 10 µM isoPLB indicates the best fit to single exponential function with a time constant of 44.3 or 21.2 msec, respectively. The decaying rate of I K(erg) evoked during the envelope-of-tail occurred in single exponential function, and the presence of isoPLB yielded an increase in the decay rate of I K(erg) evoked upon such envelope-of-tail test.

Modification by IsoPLB of the V hys Behavior of I K(erg) Activated by Inverted Double Ramp Voltage (V ramp )
Earlier reports have demonstrated the capability of I K(erg) 's V hys strength to perturb either various patterns of bursting, firing, or action potential configurations intrinsically in different types of excitable cells [31,[36][37][38][39][40][41][42][43][44]46]. Therefore, we next assessed whether and how the exposure of isoPLB could modify the V hys strength of the I K(erg) evoked with double (i.e., inverted isosceles-triangular) V ramp . In this series of experiments, we voltageclamped the tested cell at −10 mV, and through D/A conversion, a downward (forward or descending) ramp from −0 to −100 mV followed by an upward (backward or ascending) limb back to 0 mV (i.e., inverted isosceles-triangular V ramp ) was then delivered (Figure 6). Under such a maneuver, the V hys of I K(erg) in response to such a double V ramp was clearly observable; that is, the current magnitude evoked during the downward and upward limb of V ramp (i.e., the instantaneous I-V relationship of I K(erg) ) was highly distinguishable and the direction of I K(erg) over time was noticed to display a clockwise direction. For example, during the control period (i.e., absence of isoPLB), the I K(erg) amplitude taken at −80 mV during the downward ramp of double V ramp was 423 ± 19 pA (n = 8), a value that was much greater than that measured at the same level of voltage during the upward end (45 ± 6 pA; n = 8, p < 0.01). As the V ramp speed became slowed, the V hys strength activated was progressively reduced.
that is, the current magnitude evoked during the downward and upward limb of Vramp (i.e., the instantaneous I-V relationship of IK(erg)) was highly distinguishable and the direction of IK(erg) over time was noticed to display a clockwise direction. For example, during the control period (i.e., absence of isoPLB), the IK(erg) amplitude taken at −80 mV during the downward ramp of double Vramp was 423 ± 19 pA (n = 8), a value that was much greater than that measured at the same level of voltage during the upward end (45 ± 6 pA; n = 8, p < 0.01). As the Vramp speed became slowed, the Vhys strength activated was progressively reduced.
Of additional interest, during cell exposure to 3 or 10 μM isoPLB, the evolving Vhys strength responding to an inverted double Vramp was robustly lessened as the isoPLB concentration was raised ( Figure 6). For example, as the duration of the double Vramp delivered was set at 1.2 sec with a ramp speed of ±183.3 mV/sec, the value of ∆area (i.e., the difference in the area encircled by the curve in the downward and upward direction) for Vhys in the control period was calculated to be 19.5 ± 2.6 mV·nA (n = 8), while the value of ∆area in the presence of 3 or 10 μM isoPLB was measurably reduced to 10.1 ± 1.4 mV·nA (n = 8, p < 0.05) or 6.2 ± 0.8 mV·nA (n = 8, p < 0.05), respectively. Additionally, with cell exposure to isoPLB (10 μM), the further addition of PD118057 (10 μM) reversed the ∆area value to 12.3 ± 1.6 mV·nA (n = 8, p < 0.05). PD118057 has been reported to enhance IK(erg) [78]. Of additional interest, during cell exposure to 3 or 10 µM isoPLB, the evolving V hys strength responding to an inverted double V ramp was robustly lessened as the isoPLB concentration was raised ( Figure 6). For example, as the duration of the double V ramp delivered was set at 1.2 s with a ramp speed of ±183.3 mV/s, the value of ∆area (i.e., the difference in the area encircled by the curve in the downward and upward direction) for V hys in the control period was calculated to be 19.5 ± 2.6 mV·nA (n = 8), while the value of ∆area in the presence of 3 or 10 µM isoPLB was measurably reduced to 10.1 ± 1.4 mV·nA (n = 8, p < 0.05) or 6.2 ± 0.8 mV·nA (n = 8, p < 0.05), respectively. Additionally, with cell exposure to isoPLB (10 µM), the further addition of PD118057 (10 µM) reversed the ∆area value to 12.3 ± 1.6 mV·nA (n = 8, p < 0.05). PD118057 has been reported to enhance I K(erg) [78].

Mild Inhibitory Effect on Delayed-Rectifier K + Current (I K(DR) ) Produced by IsoPLB
Another set of experiments was undertaken to determine if different types of K + currents (e.g., I K(DR) ) in GH 3 cells can be modified by the presence of isoPLB. We bathed cells in a Ca 2+ -free Tyrode's solution which contained 1 µM TTX and 0.5 mM CdCl 2 , and the electrode was filled up with a K + -enriched solution. As isoPLB at a concentration of 1 µM was applied, no appreciable change in I K(DR) amplitude elicited by the step depolarization to +50 mV from a holding potential of −50 mV was demonstrated. However, one minute of exposing cells to isoPLB (10 µM) decreased the I K(DR) amplitude evoked by membrane depolarization. The average I-V relationship of the I K(DR) obtained during the control period (i.e., absence of isoPLB) or cell exposure to isoPLB (10 µM) was established and is then illustrated in Figure 7A,B. For example, with cell exposure to 10 µM isoPLB, as the tested cell was 1-s depolarized from −50 to +50 mV with a duration of 1 s, the I K(DR) measured at the end of the voltage pulse was significantly decreased to 545 ± 78 pA (n = 7, p < 0.05) from a control value of 690 ± 121 pA (n = 7). As isoPLB was removed, the current amplitude returned to 687 ± 119 pA (n = 7). Likewise, in the presence of 10 µM isoPLB, the whole-cell conductance of I K(DR) measured at the potentials ranging from +10 to +50 mV was also reduced to 7.67 ± 0.21 nS (n = 7, p < 0.05) from a control value of 8.13 ± 0.23 nS (n = 7). These results prompt us to reflect that the presence of isoPLB (10 µM) yields a mild inhibitory effect on the I K(DR) obtained in GH 3 cells.

Inability of IsoPLB to Alter Voltage-Gated Na + Current (I Na ) in GH 3 Cells
Some of the compounds which have been revealed to block I K(erg) have been noticed to be engaged in altering the magnitude and gating kinetics of I Na [33,57]. We therefore wanted to investigate whether isoPLB has any effects on the I Na residing in GH 3 cells. Here, we bathed cells in a Ca 2+ -free Tyrode's solution, in which 10 mM TEA and 0.5 mM CdCl 2 were included. The pipettes used were filled with a Cs + -containing solution, the composition of which was stated under Materials and Methods. The tested cell was depolarized from −100 to −10 mV with a duration of 30 ms to evoke I Na with a fast activating and inactivation time course during a rapid depolarizing command pulse [33,69,79]. With the exposure to 10 µM isoPLB, the peak amplitude of the I Na remained unaffected ( Figure 8A,B). The presence of 10 µM PLB also had little or no effect on the I Na . However, during continued exposure to isoPLB, the subsequent addition of either PF-04856264 (3 µM) or TTX (1 µM) was active in suppressing the I Na amplitude. PF-04856264 has been previously reported to be a synthetic blocker of Na V 1.7 channels [80]. A summary bar graph demonstrating the effectiveness of isoPLB, PLB, isoPLB plus PF-04856264, and isoPLB plus TTX in altering I K(erg) is illustrated in Figure 8B. Unlike I K(erg) , it thus appears that the I Na evoked during rapid membrane depolarization is refractory to modification by the presence of isoPLB or PLB. measured at the end of the voltage pulse was significantly decreased to 545 ± 78 pA (n = 7, p < 0.05) from a control value of 690 ± 121 pA (n = 7). As isoPLB was removed, the current amplitude returned to 687 ± 119 pA (n = 7). Likewise, in the presence of 10 μM isoPLB, the whole-cell conductance of IK(DR) measured at the potentials ranging from +10 to +50 mV was also reduced to 7.67 ± 0.21 nS (n = 7, p < 0.05) from a control value of 8.13 ± 0.23 nS (n = 7). These results prompt us to reflect that the presence of isoPLB (10 μM) yields a mild inhibitory effect on the IK(DR) obtained in GH3 cells.

Effect of IsoPLB on I K(erg) in MA-10 Leydig Tumor Cells
IsoPLB has been since reported to exercise anti-neoplastic actions in an array of cancer cells [3,[47][48][49][51][52][53][54]. Therefore, in a final set of experiments, we further wanted to test if any modifications of I K(erg) in other types of neoplastic cells (e.g., MA-10 cells) could be induced by the presence of isoPLB. The experimental protocol applied was similar to that used above in GH 3 cells. The application of isoPLB drastically reduced the peak amplitude of the I K(erg) measured over the entire voltage ranges examined. The average I-V relationship of peak I K(erg) with or without exposure to 3 µM isoPLB was constructed and is hence illustrated in Figure 9. For example, cell exposure to isoPLB at a concentration of 3 µM significantly decreased the peak I K(erg) measured at −100 mV to 318 ± 38 pA (n = 7, p < 0.05) from a control value of 615 ± 45 pA (n = 7). After isoPLB was removed, the current amplitude returned to 608 ± 41 pA (n = 7). It is evident from the results that, similar to that seen above in GH 3 cells, the sizable I K(erg) identified in MA-10 cells was sensitive to inhibition by the isoPLB presence, although MA-10 cells were not found to display the presence of I Na .
8A,B). The presence of 10 μM PLB also had little or no effect on the INa. However, during continued exposure to isoPLB, the subsequent addition of either PF-04856264 (3 μM) or TTX (1 μM) was active in suppressing the INa amplitude. PF-04856264 has been previously reported to be a synthetic blocker of NaV1.7 channels [80]. A summary bar graph demonstrating the effectiveness of isoPLB, PLB, isoPLB plus PF-04856264, and isoPLB plus TTX in altering IK(erg) is illustrated in Figure 8B. Unlike IK(erg), it thus appears that the INa evoked during rapid membrane depolarization is refractory to modification by the presence of isoPLB or PLB.

Effect of IsoPLB on IK(erg) in MA-10 Leydig Tumor Cells
IsoPLB has been since reported to exercise anti-neoplastic actions in an array of cancer cells [3,[47][48][49][51][52][53][54]. Therefore, in a final set of experiments, we further wanted to test if any modifications of IK(erg) in other types of neoplastic cells (e.g., MA-10 cells) could be induced by the presence of isoPLB. The experimental protocol applied was similar to that used above in GH3 cells. The application of isoPLB drastically reduced the peak amplitude of the IK(erg) measured over the entire voltage ranges examined. The average I-V relationship of peak IK(erg) with or without exposure to 3 μM isoPLB was constructed and is hence illustrated in Figure 9. For example, cell exposure to isoPLB at a concentration of 3 μM significantly decreased the peak IK(erg) measured at −100 mV to 318 ± 38 pA (n = 7, p < 0.05) from a control value of 615 ± 45 pA (n = 7). After isoPLB was removed, the current amplitude returned to 608 ± 41 pA (n = 7). It is evident from the results that, similar to that seen above in GH3 cells, the sizable IK(erg) identified in MA-10 cells was sensitive to inhibition by the isoPLB presence, although MA-10 cells were not found to display the presence of INa. . We measured the current amplitude at the start of each voltage pulse. The analyses with or without the addition of isoPLB measured at different levels of membrane potentials were performed with ANOVA-2 (p < 0.05).

Discussion
The important findings shown in this study are that the presence of isoPLB can regulate the magnitude, gating kinetics, and Vhys of the IK(erg) obtained in GH3 cells. However, the magnitude of the INa in these cells failed to be affected by this compound, while that of the IK(DR) was mildly suppressed. We also observed that the IK(erg) identified in MA-10 . We measured the current amplitude at the start of each voltage pulse. The analyses with or without the addition of isoPLB measured at different levels of membrane potentials were performed with ANOVA-2 (p < 0.05).

Discussion
The important findings shown in this study are that the presence of isoPLB can regulate the magnitude, gating kinetics, and V hys of the I K(erg) obtained in GH 3 cells. However, the magnitude of the I Na in these cells failed to be affected by this compound, while that of the I K(DR) was mildly suppressed. We also observed that the I K(erg) identified in MA-10 Leydig tumor cells was subjected to a block by isoPLB. The experimental observations prompted us to imply that the reduction by isoPLB or PLB of I K(erg) could potentially contribute to their anti-neoplastic actions, presuming that similar in vivo findings occur.
Earlier investigations have demonstrated the effectiveness of isoPLB in perturbing mitochondrial respiration or the mitochondria-mediated induction of reactive oxygen species in many types of neoplastic cells [10,12,15,25,26]. Mitochondrial dysfunction would be expected to decrease cytosolic ATP content, thereby resulting in changes in the activity of ATP-sensitive K + (K ATP ) channels [74,81]. However, it seems that the inhibitory effect of isoPLB or PLB on the I K(erg) magnitude seen in GH 3 cells is unlikely to be mainly due to its activity as a regulator of K ATP channels. The notable reasons can be explained as follows: (1) during whole-cell configuration in our experimental conditions, the ATP concentration of the pipette internal solution was 3 mM, a value that suffices to fully block the activity of K ATP channels [74,75], and (2) an isoPLB-induced block of I K(erg) could not be counteracted by subsequent exposure to diazoxide, an opener of K ATP channels [74,75].
It needs to be mentioned herein that PLB was able to suppress the activities of intestinal Ca 2+ -activated Cl − channels and the cystic fibrosis transmembrane conductance regulator (CFTR) or other types of Cl − channels in cancer cells [82,83]. Pituitary cells have also reported the functional expression of Cl − channels [30,64,84]. However, under our wholecell current recordings for the measurement of I K(erg) , Cl − ions inside the internal solution has been almost replaced with aspartate. Moreover, the I K(erg) magnitude measured during long-lasting step hyperpolarization was not altered by adding chlorotoxin, an inhibitor of Cl − channels [70], yet was sensitive to a block by azimilide, a blocker of I K(erg) [71]. The block by isoPLB or PLB of I K(erg) seen in GH 3 cells is unlikely to be associated with its action on the activity of Cl − channels reported recently [82,83].
In this study, as with azimilide, the presence of DTDP alone, a sulfhydryl oxidizing agent [72,73], was found to suppress I K(erg) amplitude in GH 3 cells. However, with cell exposure to DTDP, the subsequent addition of isoPLB failed to exert further suppressive effects on I K(erg) . PLB has been reported to induce free radical species in cervical cancer cells [3], as well as being an inhibitor of NADPH oxidase-4 [26], while 2-amino-1,4-naphthoquinone could induce the production of free radicals [56]. It is therefore plausible to speculate that the interaction with K erg channels accompanied by the block of I K(erg) could be shared to some extent by the DTDP and isoPLB molecules. To what extent the isoPLB-mediated block of I K(erg) is associated with the production of reactive oxygen species also remains to be further resolved.
We have shown that I K(erg) in GH 3 cells did undergo V hys behavior during the inverted isosceles-triangular V ramp , indicating that the K erg channel residing in GH 3 cells displays a clear V hys in the voltage gating closely linked to the voltage-sensor domain residing in the channel [67,68]. The I K(erg) 's V hys intrinsically in GH 3 cells reflects that a mode shift during channel activation may exist, because the voltage sensitivity of the gating charge movement relies on the previous state (or conformation) of K erg channels. Under such a scenario, as the membrane potential become negative (i.e., downward ramp of the double V ramp ), the voltage dependence of K erg channels may shift the mode of V hys to one which occurs at more negative potentials, leading to an increase in membrane repolarization. However, as the membrane potential becomes depolarized (i.e., during initiation of action potentials or the upward limb of the triangular V ramp ), the voltage dependence of I K(erg) activation would switch to less depolarized voltages with a smaller current magnitude, thereby having the tendency to increase cell excitability. Both the slowing in recovery of the I K(erg) block and the increase in the decay of I K(erg) elicited with the envelope-of-tail method also strongly support the notion that the molecule of isoPLB or PLB interacts with the open states (conformations) of the K erg channel. Under this scenario, the block by isoPLB or PLB of I K(erg) demonstrated herein would be expected to have an important impact on the discharge patterns of action potentials occurring in excitable cells.
The resulting IC 50 values required for an isoPLB-mediated block of peak and sustained I K(erg) were 18.3 or 2.4 µM, respectively. From the first-order reaction scheme, the K D value was quantitatively calculated to be 2.58 µM, a value that was noted to dovetail with the IC 50 (i.e., 2.4 µM) for isoPLB-induced inhibition of sustained I K(erg) evoked by long-lasting membrane hyperpolarization. An earlier study has shown that the isoPLB at a concentration of 2.5 µM could suppress the activity of mitochondrial respiration through a mechanism of its inhibitory action on complex IV activation [12]. Moreover, any modifications by isoPLB or PLB of I K(erg) depend not only on the concentration of isoPLB or PLB given but also on different confounding variables, including the pre-existing level of the resting potential, various discharge patterns of action potentials, and their combinations, presuming that the magnitude of I K(erg) is adequately and functionally expressed in the cells examined. Therefore, whatever the detailed mechanism of isoPLB-inhibited actions on I K(erg) which remains resolved, the block by isoPLB or PLB of I K(erg) demonstrated herein is of pharmacological relevance [35,62]. It is likely that the presence of isoPLB or PLB may induce the secretion of prolactin in vivo directly through the inhibition of I K(erg) [30,31,36,46,55,58]. However, to what extent an isoPLB-mediated block of I K(erg) affects cardiac function [32] remains to be delineated.
Since our results have shown that isoPLB and PLB have inhibitory effects on the magnitude, gating kinetics, and V hys of I K(erg) , we next investigated whether isoPLB and PLB would directly bind to the hERG protein to affect its functional activity. The hERG protein was docked with isoPLB, PLB, and the known inhibitor doxazosin, through PyRx software. The predicted binding sites of these compounds were shown in Figure 10. Doxazosin forms hydrogen bonds with residue Y616 in pore helix P1 and forms hydrophobic contacts with residues V612, L615, F619, S620, T623, and F627 in pore helix P1 and residues Y652, and F656 in helix S6. IsoPLB forms hydrogen bonds with residue N629 in pore helix P1 and forms hydrophobic contact with residues V612, T613, and Y616 in pore helix P1 and residues L589, Q592, and I593 in the segment between S5 and P1. PLB form hydrophobic contacts with residues A565, and C566 in helix S5, residues T569, I607, K610, and Y611 in the segment between S5 and P1, and residue A614 in pore helix P1. Previous studies have reported that the expression of hERG channels in a variety of tumor cells could facilitate cell proliferation [47,48,85], and doxazosin inhibits hERG currents to reduce the proliferation of tumor cells [86,87]. Since our results have shown an inhibitory effect of isoPLB and PLB on the hERG current and a predicted interaction between the hERG protein and isoPLB or PLB, isoPLB and PLB could potentially be applied to treat neoplastic cells. Due to the fact that isoPLB binds preferentially to the extracellular domain of hERG, it is likely a more effective inhibitor than doxazosin. drial respiration [12], our results suggest that the inhibitory effects of isoPLB or PLB on the magnitude, gating kinetics, and Vhys of IK(erg), appear to be obligate mechanisms. Through the ionic mechanism of their actions occurring in a concentration-, time-, state-, and hysteresis-dependent manner, it or other structurally similar compounds are anticipated to influence the functional activities or aberrant growth of different neoplastic cells, if similar in vivo results are found [9,16,29,[48][49][50][51]54,82]. IsoPLB, PLB, or other structurally related compounds [88] could be intriguing compounds useful for characterizing the Kerg channels. Figure 10. Docking results of hERG and doxazosin, isoPLB, and PLB. The protein structure of hERG was obtained from PDB (PDB ID: 5VA1) and the compound structure of doxazosin, isoPLB, and Figure 10. Docking results of hERG and doxazosin, isoPLB, and PLB. The protein structure of hERG was obtained from PDB (PDB ID: 5VA1) and the compound structure of doxazosin, isoPLB, and PLB were obtained from PubChem (compound CID: 3157, 10205, 375105). The structure of hERG amino acid 520-660 was extracted through PyMOL and then docked with three compounds through PyRx. The diagrams of interaction between hERG and compounds were generated by LigPlot+. The red arcs with spokes radiating toward the ligand represent hydrophobic contacts and green dotted lines represent hydrogen bonds.
In light of the present study, apart from the modifying effects of isoPLB on mitochondrial respiration [12], our results suggest that the inhibitory effects of isoPLB or PLB on the magnitude, gating kinetics, and V hys of I K(erg) , appear to be obligate mechanisms. Through the ionic mechanism of their actions occurring in a concentration-, time-, state-, and hysteresis-dependent manner, it or other structurally similar compounds are anticipated to influence the functional activities or aberrant growth of different neoplastic cells, if similar in vivo results are found [9,16,29,[48][49][50][51]54,82]. IsoPLB, PLB, or other structurally related compounds [88] could be intriguing compounds useful for characterizing the K erg channels.