Characterization of Perturbing Actions by Verteporfin, a Benzoporphyrin Photosensitizer, on Membrane Ionic Currents

Verteporfin (VP), a benzoporphyrin derivative, has been clinically tailored as a photosensitizer and recently known to suppress YAP-TEAD complex accompanied by suppression of the growth in an array of neoplastic cells. However, the detailed information is little available regarding possible modifications of it and its related compounds on transmembrane ionic currents, despite its growing use in clinical settings. In this study, from whole cell recordings, VP (0.3–100 μM) increased the amplitude of Ca2+-activated K+ currents (IK(Ca)) in pituitary tumor (GH3) cells in a concentration-dependent manner with an EC50 value of 2.4 μM. VP-stimulated IK(Ca) in these cells was suppressed by further addition of either paxilline, iberiotoxin, or dithiothreitol, but not by that of tobultamide or TRAM-39. VP at a concentration of 10 μM mildly suppressed the amplitude of delayed-rectifier K+ current; however, it had minimal effects on M-type K+ current. In cell-attached current recordings, addition of VP to the recording medium enhanced the activity of large-conductance Ca2+-activated K+ (BKCa) channels. In the presence of VP, additional illumination with light intensity of 5.5 mW/cm2 raised the probability of BKCa-channel openings further. Addition of VP decreased the peak amplitude of L-type Ca2+ current together with slowed inactivation time course of the current; however, it failed to modify voltage-gated Na+ current. Illumination of GH3 cells in continued presence of VP also induced a non-selective cation current. Additionally, VP increased the activity of BKCa channels in human 13-06-MG glioma cells with an EC50 value of 1.9 μM. Therefore, the effects of VP on ionic currents described herein tend to be upstream of its inhibition of YAP-TEAD complex and they are conceivably likely to contribute to the underlying mechanisms through which it and its structurally similar compounds effect the modifications in functional activities of pituitary or glial neoplastic cells, if the in vivo findings occur.

Verteporfin (VP), a benzoporphyrin derivative, has been clinically tailored as a photosensitizer and recently known to suppress YAP-TEAD complex accompanied by suppression of the growth in an array of neoplastic cells. However, the detailed information is little available regarding possible modifications of it and its related compounds on transmembrane ionic currents, despite its growing use in clinical settings. In this study, from whole cell recordings, VP (0.3-100 µM) increased the amplitude of Ca 2+ -activated K + currents (I K(Ca) ) in pituitary tumor (GH 3 ) cells in a concentration-dependent manner with an EC 50 value of 2.4 µM. VP-stimulated I K(Ca) in these cells was suppressed by further addition of either paxilline, iberiotoxin, or dithiothreitol, but not by that of tobultamide or TRAM-39. VP at a concentration of 10 µM mildly suppressed the amplitude of delayed-rectifier K + current; however, it had minimal effects on M-type K + current. In cell-attached current recordings, addition of VP to the recording medium enhanced the activity of large-conductance Ca 2+ -activated K + (BK Ca ) channels. In the presence of VP, additional illumination with light intensity of 5.5 mW/cm 2 raised the probability of BK Ca -channel openings further. Addition of VP decreased the peak amplitude of L-type Ca 2+ current together with slowed inactivation time course of the current; however, it failed to modify voltage-gated Na + current. Illumination of GH 3 cells in continued presence of VP also induced a non-selective cation current. Additionally, VP increased the activity of BK Ca channels in human 13-06-MG glioma cells with an EC 50 value of 1.9 µM. Therefore, the effects of VP on ionic currents described herein tend to be upstream of its inhibition of YAP-TEAD complex and they are conceivably likely to contribute to the underlying mechanisms through which it and its structurally similar compounds effect the modifications in functional activities of pituitary or glial neoplastic cells, if the in vivo findings occur.
VP on the level of surface membrane including ionic channels in a variety of neoplastic cells, despite its clinical approval for photodynamic therapy.
Because of the considerations described above, we sought to determine whether VP and its related compounds could exert any possible perturbations on different types of ionic currents which include Ca 2+ -activated K + current (I K(Ca) ), delayed-rectifier K + current (I K(DR) ), M-type K + current (I K(M) ), large-conductance Ca 2+ -activated K + (BK Ca ) channel, L-type Ca 2+ current (I Ca,L ), and voltage-gated Na + current (I Na ). In this study, we provide substantial evidence to unravel that VP is indeed capable of modifying membrane ionic currents in pituitary tumor (GH 3 ) cells and in glioma (13-06-MG) cells, the actions of which are apparently of clinical relevance and appear to be upstream of its inhibition at YAC-TEAD complex.
The composition of bath solution (i.e., HEPES-buffered normal Tyrode's solution) for GH 3 or 13-06-MG cells was as follows (in mM): NaCl 136.5, KCl 5.4, CaCl 2 1.8, MgCl 2 0.53, glucose 5.5, and HEPES-NaOH buffer 5.5 (pH 7.4). To measure K + currents [i.e., I K(Ca) , I K(DR), and I K(M) ], we filled the patch pipette with a solution (in mM): KCl 140, MgCl 2 1, Na 2 ATP 3, Na 2 GTP 0.1, EGTA 0.1, and HEPES-KOH buffer 5 (pH 7.2); however, in some separate set of experiments of reducing the level of intracellular Ca 2+ , EGTA concentration in the solution was changed to 10 mM. To record I K(M) , high K + -bathing solution was composed of the following (in mM): KCl 145, MgCl 2 0.53, and HEPES-KOH 5 (pH 7.4). In order to measure I Na , I Ca,L , or I NS precisely, we replaced K + ions inside the pipette solution with equimolar Cs + ions, and pH was then appropriately adjusted to 7.2 with CsOH. In order to fully suppress Na + , Ca 2+ or K + currents, TTX (1 µM), CdCl 2 (0.5 mM) or TEA (10 mM) was added to the recording medium, respectively. The pipette solution and culture medium were commonly filtered on the day of use with Acrodisc R syringe filter with 0.2 µm Supor R membrane (Pall Corp., Port Washington, NY).

Cell Culture
GH 3 pituitary tumor cells, obtained from the Bioresources Collection and Research Center ([BCRC-60015]; Hsinchu, Taiwan), were maintained in Ham's F-12 media supplemented with 15% horse serum (v/v), 2.5% fetal calf serum (v/v), and 2 mM L-glutamine (Wu et al., 2003;Lin et al., 2014;So et al., 2018). The glioblastoma multiforme cell line (13-06-MG) was kindly provided by Professor Dr. Carol A. Kruse, Department of Neurosurgery, Ronald Reagan UCLA Medical Center, LA, CA, USA. Cells were grown in high-glucose (4 g/l) Dulbecco's modified Eagle media supplemented with 10% heat-inactivated fetal bovine serum . GH 3 or 13-06-MG cells were maintained at 37 • C in a humidified environment of 5% CO 2 /95% air and sub-cultured weekly and fresh media were generally added every 2-3 days to maintain a healthy cell population. Glial cells were verified by identifying glial fibrillary acidic protein, a cytoskeletal protein. The experiments were performed after 5 or 6 days of subcultivation (60-80% confluence).

Electrophysiological Measurements
Shortly before experimentation, we dissociated the cells and then placed a few drops of cell suspension onto a custom-built recording chamber affixed on the stage of a DM-IL inverted microscope (Leica, Wetzlar, Germany). As being settled down the bottom of the chamber, cells were bathed at a room temperature of 20-25 • C in normal Tyrode's solution, the composition of which is describe above. Once cells were placed in the VPcontaining medium, the recordings were carried out under very dim room light. For illumination system, we focused visible light onto a spot on the recording chamber which covered completely the area of cells exposed to VP in the central region with an illumination intensity of ∼5.5 mW/cm 2 . We fabricated the recording pipette from Kimax-51 capillary tubes (#34500; Kimble, Vineland, NJ) using a vertical PP-83 (Narishige, Tokyo, Japan) or a horizontal P-97 Flaming/Brown (Sutter, Novato, CA) puller, and their tips were fire-polished with an MF-83 microforge (Narishige). During the measurements, the pipette with a resistance of 3-5 M , which was inserted into holder, was gently maneuvered by using a WR-98 micromanipulator (Narishige). Patch-clamp experiments were measured in either cell-attached, inside-out, or whole-cell arrangement by using an RK-400 patch-clamp amplifier (Bio-Logic, Claix, France) connected with a personal computer with 64-bit processor (Lin et al., 2004;Wu et al., 2017;So et al., 2019). Shortly before giga-seal formation was achieved, the potentials were appropriately corrected for the liquid junction potential that commonly develop at the pipette tip as the composition of the pipette solution was quite different from that in the bath. Tested compounds were applied by perfusion or added to the bath to obtain the final concentration indicated.

Data Recordings
The data comprising both potential and current traces were stored online in an ASUS VivoBook Flip-14 touchscreen laptop computer (TP412U; Taipei City, Taiwan) at 10 kHz equipped with the 12-bit Digidata 1440A interface (Molecular Devices, Inc., Sunnyvale, CA). The latter device was used for efficient analog-to-digital/digital-to-analog conversion. During the experiments, data acquisition system was electronically driven by pCLAMP 10.7 software (Molecular Devices) run under Windows 10 (Redmond, WA), and the signals were simultaneously monitored on LCD monitor (MB169B+; ASUS, Taipei, Taiwan) through a USB type-C connection. Current signals were low-pass filtered at 2 kHz with FL-4 four-pole Bessel filter (Dagan, Minneapolis, MN) to minimize background noise. Through digital-to-analog conversion, the pCLAMP-generated voltage-clamp profiles with different waveforms were applied to determine the current-voltage (I-V) relationships for different types of ionic currents such as I K(Ca) , I K(DR), or I Ca,L . When high-frequency stimuli used to elicit the cells were required, an Astro-med Grass S88X dual output pulse stimulator (Grass Technologies, West Warwick, RI) was used.

Data Analyses
To evaluate concentration-dependent effect of VP on the stimulation of I K(Ca) in GH 3 cells, each cell was depolarized to +50 mV from a holding potential of 0 mV. Current amplitude measured at the end of each depolarizing pulse during cell exposure to 100 µM VP was taken to be 100%, and those in the presence of different VP concentrations were then compared. To determine concentration-dependent stimulation of VP on BK Ca channels recorded from human 13-06-MG glioma cells, channel activity at +60 mV relative to the bath during the exposure to 100 µM VP was considered as 100%, and the channel open probabilities at different VP concentrations were compared. The mean values for concentration-dependent relation of VP on the stimulation of I K(Ca) or BK Ca channels were least-squares fitted to a modified Hill function. That is, where [VP] is the VP concentration applied, EC 50 the concentration required for half-maximal stimulation of I K(Ca) or BK Ca channels, n H the Hill coefficient, and E max VP-induced maximal stimulation of either I K(Ca) in GH 3 cells or BK Ca channels in 13-06-MG cells.
Single-BK Ca channel currents were analyzed by pClamp 10.7 software (Molecular Devices). Multi-gaussian adjustments of the amplitude distributions among channels were commonly used to determine single-channel currents. Functional independence between channels was verified by comparing the observed stationary probabilities with the values calculated according to the binomial law. The open-state probability of the channels was expressed as N·P O which can be estimated using the following equation: where N is the number of active channels in the patch examined, A 0 the area under the curve of an all-points histogram corresponding to the closed state, and A 1 -A n are the histogram areas reflecting the levels of distinct open state for 1 to n channels in the patch.

Statistical Analyses
To perform linear or non-linear (e.g., sigmoidal or exponential function) curve fitting to the data set (i.e., the goodness of fit) was appropriately utilized by using OriginPro (OriginLab, Northampton, MA) or Prism version 5.0 (GraphPad, La Jolla, CA). Data were analyzed and are plotted using OriginPro (OriginLab), and they were expressed as mean±standard error of the mean (SEM). The paired or unpaired Student's t-test, or oneway analysis of variance (ANOVA) followed by post-hoc Fisher's least-significance difference test for multiple comparisons, were implemented for the statistical evaluation of differences among means. We further used non-parametric Kruskal-Wallis test, as the assumption of normality underlying ANOVA was likely to be violated. Statistical analyses were commonly performed by using IBM SPSS R version 20.0 (IBM Corp., Armonk, NY). P < 0.05 was considered significant, unless stated otherwise.

Stimulatory Effect of VP on Ca 2+ -Activated K + (I K(Ca) ) Measured From Pituitary Tumor (GH 3 ) Cells
The whole cell configuration of the patch-clamp technique was initially carried out to evaluate any possible perturbations of VP on I K(Ca) amplitude in these cells. In the experiments designed to measure I K(Ca) , we immersed the cells in normal Tyrode's solution containing 1.8 mM CaCl 2 , and the recording pipette was filled with a solution which contained a low concentration 140 mM K + , 0.1 mM EGTA, and 3 mM ATP. As whole-cell mode was achieved, the examined cell was maintained at the level of 0 mV to inactivate other types of voltage-gated K + currents which are enriched in these cells (Stojilkovic et al., 2010;So et al., 2018), and ionic currents in response to a series of the voltage pulses between 0 and +60 mV were robustly elicited. Current amplitude was progressively increased as the test voltage became more positive (Figure 1). When extracellular Ca 2+ was removed, current amplitudes were greatly reduced; meanwhile, an increase in intracellular EGTA concentration from 0.1 to 10 mM also abolished these currents. This type of outward currents with an outwardly rectifying property has been hence regarded as I K(Ca) (Lin et al., 2004;Wu et al., 2017), the amplitudes of which become greatly small as intracellular Ca 2+ level is decreased (i.e., intracellular EGTA with 10 mM). As cells were exposed to VP, the amplitude of I K(Ca) in response to depolarizing step was considerably increased (Figures 1A,B). For example, VP at a concentration of 3 µM significantly raised I K(Ca) measured at the level of +50 mV from 184 ± 13 to 257 ± 24 pA (n = 11, P < 0.05). Washout of VP, current amplitude returned to 197 ± 14 pA (n = 10, P < 0.05). Similarly, as cells were exposed to 10 µM NS1619, an activator of BK Ca channels, I K(Ca) amplitude at +50 mV increased from 179 ± 14 to 235 ± 25 pA (n = 8, P < 0.05). Figure 1B illustrates the averaged current-voltage (I-V) relationships of I K(Ca) with or without addition of VP (3 µM).
Concentration-dependent stimulation of I K(Ca) on GH 3 cells by VP was further derived and then constructed. In these experiments, we bathed the cells in normal Tyrode's solution, the examined cell was clamped at 0 mV, and depolarizing pulse from 0 to +50 mV was then delivered. As illustrated in Figures 2A,B, the presence of different VP concentrations (0.1-100 µM) could be efficacious at raising I K(Ca) amplitude in a concentrationdependent manner. According to least-squares minimization procedure, the half-maximal concentration required for the stimulatory effect of VP on I K(Ca) amplitude was estimated to be 2.4 µM, and it at a concentration of 100 µM fully increased current amplitude elicited by voltage depolarization. The experimental results thus demonstrate that VP has a stimulatory action on I K(Ca) seen in GH 3 cells in a concentrationdependent manner.

Comparison of Effects of VP, VP Plus Tolbutamide, VP Plus TRAM-39, VP Plus Paxilline, VP Plus Iberiotoxin, and VP Plus Dithiothreitol on I K(Ca) Amplitude in GH 3 Cells
An earlier work has demonstrated that VP might disrupt mitochondrial inner membrane potential in cancer cells (Belzacq et al., 2001). On the other hand, the activity of different types of K + channels present in pituitary cells including GH 3 cells (Stojilkovic et al., 2010) might contribute synergistically to VP-mediated effect on I K(Ca) . Therefore, during cell exposure to VP, subsequent addition of different compounds including tolbutamide, TRAM-39, paxilline, iberiotoxin, and dithiothreitol was further studied to evaluate whether those agents exert any modulation on I K(Ca) stimulated by VP. As depicted in Figure 2C, subsequent application of neither tolbutamide nor TRAM-39 could attenuate VP-mediated increase of I K(Ca) amplitude; however, that of paxilline, iberiotoxin, or dithiothreitol did reduce I K(Ca) raised by VP. Tolbutamide and TRAM-39 are blockers of ATP-sensitive K + (K ATP ) and intermediated-conductance Ca 2+ -activated K + (IK Ca ) channels (Chen P. C. et al., 2018;Chen T. S. et al., 2018;Liu et al., 2019a), respectively, while paxilline and iberiotoxin could suppress the activity of large-conductance Ca 2+ -activated K + (BK Ca ) channels, respectively. Dithiothreitol is recognized as a sulfhydryl reducing agent.
Inhibitory Effect of VP on Delayed-Rectifier K + Current (I K(DR) ) in GH 3 Cells We further studied whether, in addition to I K(Ca) , another different types of K + currents [e.g., I K(DR) ] could be modified by the presence of VP. In these experiments, cells were bathed in Ca 2+ -free Tyrode's solution containing 1 µM TTX and 0.5 mM CdCl 2 and we filled the recording pipette by using K + -containing solution. Addition of 3 µM VP was not found to modify I K(DR) amplitude significantly. However, as GH 3 cells were exposed to 10 µM VP, the I K(DR) amplitudes elicited in response to stepwise depolarizing voltages were progressively decreased (Figures 3A,B). Figure 3B illustrates averaged I-V relationships of I K(DR) measured at the end of each depolarizing pulse, as the results were obtained in the absence and presence of 10 µM VP. For example, as the examined cells were depolarized from −50 to +40 mV, addition of 10 µM VP diminished I K(DR) amplitude from 1,382 ± 145 to 1,114 ± 138 pA (n = 13, P < 0.05). After washout of the drug, current amplitude returned to 1,328 ± 141 pA (n = 11, P < 0.05). However, no modification of I K(DR) inactivation time course in response to membrane depolarization was demonstrated in the presence of 10 µM VP. Therefore, these results showed that VP at a concentration of 10 µM mildly suppressed the amplitude of I K(DR) in GH 3 cells.
Failure of VP to Modify M-Type K + Current (I K(M) ) Recorded From GH 3 Cells In addition to I K(DR) , another type of K + current [i.e., I K(M) ] detected in GH 3 cells (Sankaranarayanan and Simasko, 1996;Stojilkovic et al., 2010;So et al., 2019) was further designed to evaluate whether the presence of VP has any modifications on this type of K + current. To amplify I K(M) , we bathed cells in high-K + (145 mM), Ca 2+ -free solution, the composition of which is described in Materials and Methods. In these experiments, the examined cell held in voltage clamp at −50 mV with a long-duration depolarizing step to −10 mV was able to generate a slowly activating K + inward current followed by a largely deactivating current, namely I K(M) , as described previously (Sankaranarayanan and Simasko, 1996;Chen T. S. et al., 2018;Liu et al., 2019b;So et al., 2019). As illustrated in Figure 4, under our experimental conditions, we were unable to find out that the presence of 10 µM VP had any measurable effect on I K(M) amplitude [141 ± 19 pA [in the control] vs. 140 ± 18 pA [in the absence of 10 µM VP], n = 11, P > 0.05]. However, in continued presence of 10 µM VP, further application of 3 µM pioglitazone was effective at suppressing I K(M) , as evidenced by the results showing that, in continued presence of 10 µM VP, further application of 10 µM pioglitazone substantially decreased I K(M) amplitude to 78 ± 12 pA (n = 11, P < 0.05). Therefore, the data prompted us to indicate that distinguishable from I K(Ca) or I K(DR) , the I K(M) observed in GH 3 cells was relatively resistant to modification by VP, though it was potently suppressed by pioglitazone (Chen T. S. et al., 2018).

Effects of VP on the Activity of Bk ca Channels in GH 3 Cells
The results from our whole-cell experiments reflected that I K(Ca) described above could be K + flux through the largeconductance subtype of K Ca channels, because VP-induced increase in I K(Ca) was effectively suppressed by iberiotoxin or paxilline, yet not by TRAM-39. Therefore, in attempts to ascertain how VP interacts pertinently with ion-channel activity to modify I K(Ca) , the effects of VP on BK Ca -channel activity was . The examined cell was depolarized from 0 to +50 mV and current amplitude at the end of each depolarizing step was measured. As cells were exposed to 100 µM VP, I K(Ca) amplitude was taken to be 100%, and those at different VP concentrations were then compared. The values for EC 50 and the Hill coefficient were 2.4 µM and 1.1, respectively. Non-linear smooth line was least-squares fitted to a modified Hill function described in Materials and Methods. (C) Summary bar graph showing the effect of VP, VP plus tolbutamide, VP plus TRAM-39, VP plus paxilline, VP plus iberiotoxin, and VP plus dithiothreitol. Each bar showing I K(Ca) amplitude at the level of +60 mV indicates the mean ± SEM (n = 11-14). *Significantly different from control (P < 0.05) and **significantly different from VP (3 µM) alone group (P < 0.05).
also investigated in cell-attached or inside-out configuration. In cell-attached current recordings, cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl 2 , the activity of BK Ca channels measured at +60 mV relative to the bath can be readily detected in these cells. In particular, when VP (3 µM) was applied to the bath medium, the probability of channel openness was drastically raised (Figures 5A,B). The open probability of BK Ca channels maintained at +60 mV in the control was found to be 0.014 ± 0.004 (n = 13). One minute after addition of VP (3 µM) to the bath, the channel open probability was significantly increased to 0.024 ± 0.007 (n = 13, P < 0.05). The channel activity was reduced to 0.018 ± 0.005 (n = 12) after washout of the agent. However, the amplitude of single-channel currents remained unaltered in the presence of VP. The single-channel conductance of BK Ca channels in control cells was 146 ± 8 pS (n = 12), a value that did not differ significantly from that (147 ± 13 pS, n = 12, P > 0.05) obtained in the presence of VP (3 µM). Moreover, in continued presence of 3 µM VP, further exposure to illumination with light intensity of 5.5 mW/cm 2 increased channel open probability further to 0.047 ± 0.011 (n = 11, P < 0.01). Subsequent addition of 1 µM paxilline reduced channel activity significantly (Figure 5B), as evidenced by a significant reduction in the channel open probability to 0.017 ± 0.005 (n = 11, P < 0.05).

Effect of VP, VP Plus GMQ, and VP PF573228 on BK Ca -Channel Activity in GH 3 Cells
We also evaluated whether VP could exert any perturbation on BK Ca channels in inside-out patch of the cell. Cells were immersed in high K + solution containing 1 µM Ca 2+ , and the experiments were conducted in inside-out current recordings. As shown in Figure 6, when the excised patch was maintained at +60 mV, the addition of 3 µM VP to the intracellular leaflet of the inside-out patch did not modify the channel open probability. However, in continued presence of VP, further addition of either 2-guanidine-4-methylquinazoline (GMQ; 3 µM) or PF573228 (3 µM) was effective at raising the probability of BK Ca channels. GMQ or PF573228 was previously reported to activate BK Ca channels (So et al., 2011(So et al., , 2018. The results thus showed the inability of VP to alter the probability of BK Ca -channel openings recorded from the excised patch of GH 3 cells.

Suppressive Effect of VP on L-Type Ca 2+ Current (I Ca,L ) in GH 3 Cells
Whether VP exerts any perturbations on voltage-gated I Ca,L in these cells was further studied, because any modifications in I Ca,L magnitude can modify the level of intracellular Ca 2+ , thereby influencing either the activity of BK Ca channels or FIGURE 3 | Effects of VP on delayed-rectifier K + current (I K(DR) ) in GH 3 cells. In these experiments, we bathed cells in Ca 2+ -free Tyrode's solution containing 1 µM tetrodotoxin (TTX), and whole-cell currents were recorded by filling the pipette with K + -containing solution. The examined cell was held at −50 mV and a series of depolarizing steps ranging between −50 and +40 mV was applied [indicated in the uppermost part of (A)]. (A) Superimposed I K(DR) traces obtained during the control (upper) and cell exposure to 10 µM VP (lower). Arrowhead in each panel represents the zero-current level. (B) Averaged I-V relationships for I K(DR) obtained with or without the addition of 10 µM VP (mean ± SEM; n = 13 for each point). Current amplitudes were measured at the end of each depolarizing step. : control; : in the presence of 10 µM VP.
I K(Ca) amplitude (Wu et al., 1998a(Wu et al., ,b, 2003Stojilkovic et al., 2010). As cells were exposed to different VP concentrations the peak amplitude of I Ca,L elicited in response to single depolarizing step was progressively decreased (Figures 7A,B). The peak amplitude of I Ca,L by membrane depolarization was suppressed by nifedipine (3 µM) and enhanced by Bay K 8644 (3 µM) (data not shown). Moreover, the addition of 3 µM VP suppressed the peak amplitude of I Ca,L from 426 ± 22 to 267 ± 14 pA (n = 12, P < 0.05). Concomitant with this, the inactivation time constant of I Ca,L became slowed ( Figure 7C). The value for the slow component of I Ca,L inactivation time constant (τ inact(s) ) was prolonged to 63.7 ± 7.2 ms from a control value of 30.9 ± 5.6 ms (n = 12, P < 0.05). However, the fast component for the inactivation time constant of I Ca,L did not differ significantly between the absence and presence of 3 µM VP.
The results indicate that the presence of VP is capable of altering the amplitude and inactivation time course of I Ca,L elicited in response to rapid membrane depolarization.

Stimulatory Effect of VP on Non-selective Cation Current (I NS ) in GH 3 Cells
In another set of experiments, we tested whether VP has any effects on I NS . The results showed that addition of VP (3 µM) did not have any effect on ion currents measured at the level of −50 mV. However, in continued presence of VP, as light illumination with intensity of 5.5 mW/cm 2 was further applied, an inward current was progressively induced as the examined cell was voltage-clamp held at the level of −50 mV ( Figure 7D). Similar findings were observed in eight different GH 3 cells examined. As bathing solution was replaced with NMDG + solution, this current could still be induced in the presence of VP plus light exposure. However, chlorotoxin (1 µM), an inhibitor of Cl − channels, did not have any effects on photosensitized VP-induced inward currents. Therefore, consistent with previous observations reported from frog heart cells (Tarr et al., 1994), the inward currents seen in these cells exhibited to be the non-selective, yet not Cl − ion-specific, nature of the ionic conductance.

Inability of VP to Modify Voltage-Gated Na + Current (I Na )
Another important type of voltage-gated ionic current (i.e., I Na ) was also further examined to evaluate the possible modification of VP on this inward current. However, we were unable to detect any measurable change in the amplitude or gating of I Na elicited by brief step depolarization (Figure 8). For example, the maximal peak amplitude of I Na in the control was 2.52 ± 0.17 nA (n = 12), a value which did not differ significantly from that during cell exposure to 3 µM VP [2.52 ± 0.18 nA [n = 12, P > 0.05]].
In continued presence of 3 µM VP, subsequent addition of either A-803467 (3 µM) or A-887826 (3 µM) was potent in suppressing the peak I Na in GH 3 cells (Figure 8); however, no modification in I Na inactivation in the presence of these two agents was demonstrated. A-803467 or A-887826 was previously reported to inhibit Na V 1.8-encoded currents potently (Rush and Cummins, 2007;Zhang et al., 2010).

Effect of VP on Bk ca -Channel Activity in Human 13-06-MG Glioma Cells
VP has been recently reported to inhibit the proliferation of glioma cells Eales et al., 2018;Pellosi et al., 2019). In a final series of experiments, we therefore intended to investigate whether the presence of VP is also able to modulate ionic currents in another types of neoplastic cells (e.g., malignant glioma cells). As depicted in Figures 9A,B, in cell-attached measurements, the activity of BK Ca channels in 13-06-MG glioma cells was robustly detected as described previously Liu et al., 2015). Moreover, addition of VP to the bath increased the probability of BK Ca -channel openings in these cells. For example, as the cells were maintained at +60 mV relative to the bath, the exposure to 3 µM VP significantly elevated the channel open probability from 0.018 ± 0.007 to 0.038 ± 0.011 (n = 12, P < 0.05). Therefore, similar to that of GH 3 cells described above, BK Ca -channel activity existing in 13-06-MG cells was indeed found to be sensitive to stimulation by VP. Apart from the inhibition of YAP-TEAD complex, VPinduced inhibition of cell proliferation in glioma (Eskelin et al., 2008;Eales et al., 2018) could, to some extent, be linked to its stimulatory action on BK Ca -channel activity.

DISCUSSION
The present investigation discloses important findings that VP produced a stimulatory effect on Ca 2+ -activated K + current (I K(Ca) ) in pituitary GH 3 cells; however, it mildly depressed I K(DR) amplitude with no modification on I Na or I K(M) . The stimulatory effect on I K(Ca) caused by VP was presumably related to changes in the level of intracellular Ca 2+ concentrations. The results showing that removal of extracellular Ca 2+ suppressed VP-induced increase in I K(Ca) also suggest that extracellular Ca 2+ and internal Ca 2+ release both contribute to the increase in the amplitude of I K(Ca) in these cells. The EC 50 value for either VP-induced stimulation of I K(Ca) observed either in pituitary GH 3 cells or increase in BK Cachannel activity in 13-06-MG glioma cells was estimated to be 2.4 or 1.9 µM, respectively. These values are quite close to those that are either therapeutically achievable or required for the inhibitory actions on YAP-TEAD complex in different types of neoplastic cells (Faustino et al., 1997;Zhou et al., 2006;Feng et al., 2016;Gibault et al., 2016Gibault et al., , 2017Chen and Hu, 2017). As VP stimulated the I K(Ca) amplitude within a few minutes in GH 3 cells, it is therefore reasonable to elaborate that there is a conceivable link between the FIGURE 5 | Stimulatory effect of VP on the activity of large-conductance Ca 2+ -activated K + (BK Ca ) channels measured from on-cell patch recordings of GH 3 cells. In these cell-attached current recordings, cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl 2 and channel activity was measured by filling the pipette with K + -containing solution. (A) BK Ca channel activity measured at the holding potential of +60 mV relative to the bath. Panel a is control (i.e., in the absence of light and photosensitizer VP), while panels b and c were obtained in the presence of 3 µM VP and 3 µVP plus illumination with a light intensity of 5.5 mW/cm 2 , respectively. The lower parts in each panel indicate the expanded traces recorded from the uppermost part. Note that channel opening causes an upward defection. (B) Summary bar graph showing the effects of VP and VP plus illumination on the probability of BK Ca channels that would be open (mean ± SEM; n = 11-13 for each bar). *Significantly different from control (P < 0.05), † significantly different from control (P < 0.01), and **significantly different from VP (3 µM) plus illumination group (P < 0.01).
FIGURE 6 | The activity of BK Ca channels produced by VP, VP plus GMQ, or VP plus PF573228 under inside-out current recordings. In these experiments, cells were bathed in high-K + solution which contained 1 µM Ca 2+ , the excised patch was maintained at +60 mV, and tested compound was applied to the bath.  anti-neoplastic effects of this compound and its stimulatory actions on I K(Ca) , before it passes through the membrane (Marks et al., 2000). By using rose bengal, a natural polyphenolic compound known to be another type of photosensitizer, previous studies have demonstrated that this compound challenged with or VP plus A-887826 on the peak amplitude of I Na (mean ± SEM; n = 11 for each bar). The peak I Na amplitude was measured at the beginning of short depolarizing pulse from a holding potential of −80 mV. *Significantly different from control (P < 0.05).
without photosensitization was able to modify membrane ionic current in GH 3 cells along with mild damage on cell growth, although these cells were described to be relatively more resistant to photosensitized modification than other type of cells (Tarr et al., 1994;Tarr, 1998, 2001). Similarly, findings from this study extended the observations and showed that VP was capable of stimulating I K(Ca) in a concentration-dependent manner in GH 3 cells. The illumination of VP could induce a non-selective cation current (Tarr et al., 1994), which appeared to be associated with the production of reactive oxygen species, and, in turn, increase the amplitude of I K(Ca) further in GH 3 cells (Valenzeno and Tarr, 1998;DeRosa and Crutchley, 2002).
To what extent VP with or without illumination either modifies membrane potential or senses the photochemical modification remains to be further studied. It is also interesting to examine whether there are any other molecular target(s) which can regulate the VP-induced currents.
An earlier study has notably reported the capability of VP to dissipate mitochondrial inner transmembrane potential, hence decreasing the production of intracellular ATP (Belzacq et al., 2001). The pipette solution used in our whole-cell experiments contained 3 mM ATP; in particular, the amount of intracellular ATP is sufficient to fully suppress the activity of ATP-dependent K + (K ATP ) channels . Furthermore, VP-induced increase of outward currents observed in GH 3 cells was unable to be reversed by subsequent application of either tolbutamide, a blocker of K ATP channels, or TRAM-39, an inhibitor of IK Ca channels Chen P. C. et al., 2018;Chen T. S. et al., 2018;Liu et al., 2019a). As such, VP-induced increase in K + outward currents conceivably is not associated with the activation of K ATP or IK Ca channels. Alternatively, in continued presence of VP, the ability of subsequent addition of dithiothreitol, a sulfhydryl reducing agent, to reverse VP-stimulated I K(Ca) seems to lend credence to suggest a possible link to VP-induced production of free oxygen species (DeRosa and Crutchley, 2002;Morishita et al., 2016;Baskaran et al., 2018;Eales et al., 2018;Kim et al., 2018).
In our cell-attached current recordings, addition of VP into the bath increased the activity of BK Ca channels significantly with no clear change in the single-channel conductance. These results can be stated as meaning that VP-stimulated channel activity results largely from the availability of intracellular Ca 2+ , despite inability of the VP molecules to act within the BK Cachannel's central pore. However, VP-mediated activation of BK Ca channels found in GH 3 cells actually did not depend on increased availability of intracellular Ca 2+ owing to elevated Ca 2+ flux through voltage-gated Ca 2+ channels, given the present results showing that VP effectively suppressed the amplitude of I Ca,L in GH 3 cells. Additionally, in inside-out current recordings, as VP was applied to the intracellular surface of the excised patch, little or no change in the activity of BK Ca channels was demonstrated; however, subsequent addition of GMQ or PF573228, known to stimulate BK Ca channels, was effective at raising the channel open probability (So et al., 2011(So et al., , 2018. Notably, it has been also demonstrated that BK Ca channels are functionally expressed in either retinal pigment epithelial cells, vascular endothelial cells, or amniotic fluid-derived stem cells Schmidt-Erfurth and Hasan, 2000;Sheu et al., 2005;Hua et al., 2014;Hu et al., 2015;Liu et al., 2015Liu et al., , 2018Liu et al., , 2019a. Taking this into account, to what extent VP-induced changes in BK Cachannel activity existing in these cells are intimately connected with its actions on sensory retina or pituitary adenoma during photodynamic therapy (Marks et al., 2000;Rahimipour et al., 2003;Rosenblatt et al., 2005;Hu et al., 2015;Nemes et al., 2016;Liu et al., 2018;Min et al., 2018;Iacono et al., 2019) remains to be imperatively investigated.
As being administrated, the VP molecules need to enter into cytosol or even nucleus, before being destined for having an interaction with YAP-TEAD complex (Brodowska et al., 2014;Feng et al., 2016;Gibault et al., 2016Gibault et al., , 2017Abe et al., 2018;Qin et al., 2019); hence, they must pass through surface membrane before entering the cell interior. From findings in this study, it is therefore tempting to speculate that the perturbations on membrane ion currents caused by VP would be required to precede its subsequent actions through suppression of YAP-TEAD complex. Moreover, another important determinants needed to be considered are the VP dose and the intensity of illumination, since the stimulatory effect of VP on I K(Ca) could be highly related to generation of reactive oxygen species (Morishita et al., 2016;Baskaran et al., 2018;Eales et al., 2018;Kim et al., 2018). The sensitization sites for the modification caused by the VP molecules are thought to be probably at an intramembranous location near the inner surface. Upon cell exposure to VP particularly along with long-term light exposure, the reactive oxygen species would be excessively produced, and resultant byproducts of oxidative metabolism can then interact FIGURE 9 | Stimulatory effect of VP on BK Ca -channel activity recorded from human 13-06-MG glioma cells. In this series of experiments, we bathed cells in normal Tyrode's solution and filled the recording pipette by using high K + -containing solution. (A) Representative BK Ca -channel currents taken under a holding potential of +60 mV relative to the bath. a: control; b: 1 µM VP; c: 3 µM VP. (B) Concentration-dependent relationship of VP effect on the probability of BK Ca -channel openings in 13-06-MG cells (mean ± SEM; n = 11 for each point). The examined cell was held at +60 mV relative to the bath, and the channel open probability during the exposure to 100 µM VP was taken to be 100%. The smooth line represents the least-squares fit of the data points to the sigmoidal Hill equation as described in Materials and Methods. The values of EC 50 , Hill coefficient, and maximal percentage increase of channel activity were estimated to be 1.9 µM, 1.1, and 100%, respectively.
with membrane phospholipids and proteins that constitute ion transport pathways such as BK Ca channels. Lastly, It will be worthy of being investigated regarding to what extent VPmediated modifications on membrane ion channels reported herein is closely linked to the inhibition of VAP-TEAD complex followed by changes in proliferation or differentiation of different types of neoplastic or stem cells, given that the fact that such photosensitizers as sensitized photochemical modification in an array of tumors, including pituitary tumors and gliomas, have been widely utilized (Marks et al., 2000;Brown et al., 2004;Solban et al., 2006;Triesscheijn et al., 2006;Cole et al., 2008;Brodowska et al., 2014;Valero et al., 2015;Morishita et al., 2016;Nemes et al., 2016;Chen and Hu, 2017;Baskaran et al., 2018;Liao et al., 2018;Mulder et al., 2018;Chen et al., 2019;Li et al., 2019;Pellosi et al., 2019;Qin et al., 2019;Zhang et al., 2019). Alternatively, the efficacy of VP in inhibiting YAP/TEAD interaction was currently noted to remain debated (Lui et al., 2019). Regardless of the detailed mechanism of VP actions, it is pertinent to point out that VP-induced modifications on membrane ion channels shown herein should not be ignored and is conceivably responsible for effecting its therapeutic or adverse actions (Marks et al., 2000;Abe et al., 2018;Min et al., 2018;Iacono et al., 2019Isildak et al., 2019.

DATA AVAILABILITY
The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

ACKNOWLEDGMENTS
This study that led to the composition of this manuscript was supported in part by National Cheng Kung University (D106-35A13, D107-F2519, and NCKUH-10709001 to S-NW), Tainan City, Taiwan. The authors would like to acknowledge the technical assistance of Kaisen Lee for helping earlier work in this study. S-NW received a Talent Award for the Outstanding Researchers from Ministry of Education, Taiwan.