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High Efficacy by GAL-021: A Known Intravenous Peripheral Chemoreceptor Modulator that Suppresses BKCa-Channel Activity and Inhibits IK(M) or Ih - PubMed

  • ️Wed Jan 01 2020

High Efficacy by GAL-021: A Known Intravenous Peripheral Chemoreceptor Modulator that Suppresses BKCa-Channel Activity and Inhibits IK(M) or Ih

Te-Ling Lu et al. Biomolecules. 2020.

Abstract

: GAL-021 has recently been developed as a novel breathing control modulator. However, modifications of ionic currents produced by this agent remain uncertain, although its efficacy in suppressing the activity of big-conductance Ca2+-activated K+ (BKCa) channels has been reported. In pituitary tumor (GH3) cells, we found that the presence of GAL-021 decreased the amplitude of macroscopic Ca2+-activated K+ current (IK(Ca)) in a concentration-dependent manner with an effective IC50 of 2.33 μM. GAL-021-mediated reduction of IK(Ca) was reversed by subsequent application of verteporfin or ionomycin; however, it was not by that of diazoxide. In inside-out current recordings, the addition of GAL-021 to the bath markedly decreased the open-state probability of BKCa channels. This agent also resulted in a rightward shift in voltage dependence of the activation curve of BKCa channels; however, neither the gating charge of the curve nor single-channel conductance of the channel was changed. There was an evident lengthening of the mean closed time of BKCa channels in the presence of GAL-021, with no change in mean open time. The GAL-021 addition also suppressed M-type K+ current with an effective IC50 of 3.75 μM; however, its presence did not alter the amplitude of erg-mediated K+ current, or mildly suppressed delayed-rectifier K+ current. GAL-021 at a concentration of 30 μM could also suppress hyperpolarization-activated cationic current. In HEK293T cells expressing α-hSlo, the addition of GAL-021 was also able to suppress the BKCa-channel open probabilities, and GAL-021-mediated suppression of BKCa-channel activity was attenuated by further addition of BMS-191011. Collectively, the GAL-021 effects presented herein do not exclusively act on BKCa channels and these modifications on ionic currents exert significant influence on the functional activities of electrically excitable cells occurring in vivo.

Keywords: Ca2+-activated K+ current; GAL-021; M-type K+ current; large-conductance Ca2+-activated K+ channel; pituitary tumor cells; -hSlo.

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Conflict of interest statement

The authors declare that they have no potential conflict of interest that could be perceived as prejudicing the impartiality of the research presented herein.

Figures

Figure 1
Figure 1

Suppressive effects of GAL-021 on the magnitude of Ca2+-activated K+ current (IK(Ca)) recorded from GH3 pituitary tumor cells. In this set of whole-cell current recordings, we immersed cells in normal Tyrode’s solution containing 1.8 mM CaCl2, and the recording pipette used was filled with a K+-containing solution. Current traces are illustrated under voltage-clamp conditions during voltage pulses from 0 to +80 mV in 10-mV increments from a holding potential of 0 mV. (A) Superimposed IK(Ca) traces elicited during a series of voltage steps (indicated in the uppermost part) obtained under control conditions (upper panel), that is, when GAL-021 was not present, and during cell exposure to 1 μM GAL-021 (middle panel) or 3 μM GAL-021 (lower panel). Arrowhead in each panel depicts the zero-current level, whereas calibration mark in the right lower corner applies to all current traces illustrated. (B) Averaged current-voltage (I-V) relationships of IK(Ca) obtained in the control, during the exposure to 1 or 3 μM GAL-021, and after the washout of GAL-021. Each data point represents the mean ± SEM (n = 7 to 9). (C) Concentration-response relationship for GAL-021-mediated suppression of IK(Ca) amplitude (mean ± SEM and n = 8 to 10 for each data point). Each cell was depolarized by 300 ms long voltage step from a holding potential of 0 mV to +50 mV, and current amplitudes at the end of each depolarizing step with or without addition of different GAL-021 concentrations (0.1 to 30 μM) were measured. Continuous line in which data points were overwritten shows least-squares fit of the Hill equation (detailed in the text). The estimated values for IC50 and the Hill coefficient were 2.33 μM and 1.2, respectively.

Figure 2
Figure 2

(A) Comparisons of effects of GAL-021, GAL-021 plus diazoxide, GAL-021 plus verteporfin, and GAL-021 plus ionomycin on IK(Ca) amplitude recorded from GH3 cells. During these measurements, current amplitude was taken at the end of 300 ms voltage step from a holding potential of 0 mV to +50 mV. Current traces labeled 1 in (A) are controls, and those labeled 2 were taken during cell exposure to 3 μM GAL-021, while those labeled 3 were obtained in the presence of 3 μM GAL-021 plus 10 μM diazoxide (upper panel), or in the presence of 3 μM GAL-021 plus 10 μM verteporfin (lower panel). The uppermost part indicates the voltage protocol applied, arrowhead in each panel shows the zero-current level, and calibration mark in the right lower part in (A) applies to all current traces. (B) Summary bar graph showing effects of GAL-021, GAL-021 plus diazoxide, GAL-021 plus verteporfin, and GAL-021 plus ionomycin on IK(Ca) amplitude. Current amplitudes were recorded and then measured at the end of each depolarizing pulse from 0 to +50 mV, and each bar represents the mean ± SEM (n = 7 to 9). *, Significantly different from control (p < 0.05) and †, significantly different from GAL (3 μM) alone group (p < 0.05). GAL, 3 μM GAL-021; Diaz, 10 μM diazoxide; VP, 10 μM verteporfin; and Iono, 10 μM ionomycin.

Figure 3
Figure 3

Suppressive effect of GAL-021 on the activity of BKCa channels identified in GH3 cells. In these experiments, cells were immersed in high K+ solution containing 0.1 μM Ca2+, inside-out current recordings were performed, and channel activity was measured at the level of +60 mV. The upper or lower part in (A) shows original channel current traces or the probability of channel openings, respectively. Horizontal bar indicates the addition of 10 μM GAL-021 into the bath. The left (control) or right (during the exposure to GAL-021) current traces in the lowest part of (A) corresponds to the expanded records from dashed boxes in the upper part. Channel-opening event is denoted as an upward deflection (i.e., outward current). Of note, there is a progressive decrease in the channel open probability during the exposure to GAL-021 (10 μM). (B) Summary bar graph showing effect of GAL-021, GAL-021 plus cilostazol, and GAL-021 plus 9-phenanthrol on the probability of BKCa-channel openings (mean ± SEM and n = 6 to 8 for each bar). Inside-out current recordings were taken and the open-state probability of the channel was measured at +60 mV. * Significantly different from control (p < 0.05); **, significantly different from control (p < 0.01); † significantly different from GAL-021 (3 μM) alone group (p < 0.05); and ‡, significantly different from GAL-021 (10 μM) alone group (p < 0.05). Cil, 10 μM cilostazol and GAL, GAL-021.

Figure 3
Figure 3

Suppressive effect of GAL-021 on the activity of BKCa channels identified in GH3 cells. In these experiments, cells were immersed in high K+ solution containing 0.1 μM Ca2+, inside-out current recordings were performed, and channel activity was measured at the level of +60 mV. The upper or lower part in (A) shows original channel current traces or the probability of channel openings, respectively. Horizontal bar indicates the addition of 10 μM GAL-021 into the bath. The left (control) or right (during the exposure to GAL-021) current traces in the lowest part of (A) corresponds to the expanded records from dashed boxes in the upper part. Channel-opening event is denoted as an upward deflection (i.e., outward current). Of note, there is a progressive decrease in the channel open probability during the exposure to GAL-021 (10 μM). (B) Summary bar graph showing effect of GAL-021, GAL-021 plus cilostazol, and GAL-021 plus 9-phenanthrol on the probability of BKCa-channel openings (mean ± SEM and n = 6 to 8 for each bar). Inside-out current recordings were taken and the open-state probability of the channel was measured at +60 mV. * Significantly different from control (p < 0.05); **, significantly different from control (p < 0.01); † significantly different from GAL-021 (3 μM) alone group (p < 0.05); and ‡, significantly different from GAL-021 (10 μM) alone group (p < 0.05). Cil, 10 μM cilostazol and GAL, GAL-021.

Figure 4
Figure 4

Effect of GAL-021 on mean open (A) and closed (B) times of BKCa channels in GH3 cells. Inside-out current recordings were made in these experiments and the potential was maintained at +60 mV. Cells were immersed in high-K+ solution which contained 0.l μM Ca2+. The open- and closed-time histograms under control conditions (i.e., GAL-021 is not present) are illustrated in the left side of each panel, while those acquired during the exposure to 3 μM GAL-021 are in the right side. Of note, the abscissa and ordinate in each panel show the scale of apparent open- or closed-time histograms (msec, in a logarithmic scale) and the square root of the event numbers (n), respectively. Control data were acquired from measurements of 361 channel openings with a total record time of 1 min, whereas those taken in the presence of GAL-021 were taken from 333 channel openings with a total recording time of 2 min. Nonlinear continuous line in each histogram was least-squares fitted by single- or two-exponential function, while the broken line(s) in each lifetime distribution are pointed at the values of the time constants in open or closed state(s). Of note, the fast and slow components in mean closed time of the channel became prolonged in the presence of 3 μM GAL-021, despite its inability to modify mean open time.

Figure 5
Figure 5

The voltage-dependent effect of GAL-021 on the activity of BKCa channels identified in GH3 cells. The experiments were conducted with symmetrical K+ concentration, and we filled the electrode with K+-containing solution. Under inside-out current recordings, the potential was maintained at +60 mV and bath medium contained 0.1 μM Ca2+. (A) Failure of GAL-021 to modify the single-channel conductance of BKCa channels. The voltage ramp pulses from 0 to +120 mV with a duration of 1 sec were used to measure single-channel conductance taken with or without GAL-021 addition. The straight broken line with a reversal potential of 0 mV represents the I-V relations of the channels in the absence (upper) or presence (lower) of GAL-021 (3 μM). (B) Effect of GAL-021 on the sigmoid activation kinetics of BKCa channels. The currents illustrated were activated by applying the upsloping ramp pulses from +20 to +120 mV with a duration of 1 sec. The continuous lines showed Boltzmann fits of the data yielding 66.8 mV for the control (i.e., GAL-021 was not present) and 79.3 mV obtained as the detached patch was exposed to 3 μM GAL-021.

Figure 5
Figure 5

The voltage-dependent effect of GAL-021 on the activity of BKCa channels identified in GH3 cells. The experiments were conducted with symmetrical K+ concentration, and we filled the electrode with K+-containing solution. Under inside-out current recordings, the potential was maintained at +60 mV and bath medium contained 0.1 μM Ca2+. (A) Failure of GAL-021 to modify the single-channel conductance of BKCa channels. The voltage ramp pulses from 0 to +120 mV with a duration of 1 sec were used to measure single-channel conductance taken with or without GAL-021 addition. The straight broken line with a reversal potential of 0 mV represents the I-V relations of the channels in the absence (upper) or presence (lower) of GAL-021 (3 μM). (B) Effect of GAL-021 on the sigmoid activation kinetics of BKCa channels. The currents illustrated were activated by applying the upsloping ramp pulses from +20 to +120 mV with a duration of 1 sec. The continuous lines showed Boltzmann fits of the data yielding 66.8 mV for the control (i.e., GAL-021 was not present) and 79.3 mV obtained as the detached patch was exposed to 3 μM GAL-021.

Figure 6
Figure 6

Inhibition by GAL-021 of M-type K+ current (IK(M)) in GH3 cells. In this set of experiments, to amplify the current magnitude, cells were immersed in high-K+, Ca2+-free solution, the examined cell was maintained at −50 mV and, once whole-cell recordings were achieved, the 1 sec long step depolarization to −10 mV was applied. (A) Superimposed IK(M) traces recorded in the control (a), and during the exposure to 10 μM GAL-021 (b), or to 10 μM GAL-021 plus 10 μM flupirtine (c). (B) Summary bar graph showing effects of GAL-021, XE991, dexmedetomidine, and GAL-021 plus flupirtine on IK(M) amplitude (mean ± SEM and n = 6–9 for each bar). Current amplitudes were measured at the end of each depolarizing command pulse from a holding potential of −50 mV to −10 mV. DEX, 10 μM dexmedetomidine; Flu, 10 μM flupirtine; and GAL, 10 μM GAL-021. *, Significantly different from control (p < 0.05) and †significantly different from GAL-021 (10 μM) alone group (p < 0.05). (C) Concentration-response curve for the GAL-021-induced suppression of IK(M) (mean ± SEM and n = 6 to 8 for each data point). The smooth sigmoidal curve was fitted with the Hill equation (detailed under Materials and Methods). The IC50 value was computed to be 3.75 μM and the Hill coefficient was 1.1.

Figure 7
Figure 7

Mild inhibitory effect of GAL-021 on delayed-rectifier K+ current (IK(DR)) in GH3 cells. In these experiments, we bathed cells in Ca2+-free, Tyrode’s solution which contained 1 μM tetrodotoxin and 0.5 mM CdCl2. The holding potential was −50 mV and clamp pulses to a series of voltage steps ranging between –50 and +50 mV with a duration of 1 sec (as indicated in the uppermost part of (A) were applied. (A) Representative IK(DR) traces recorded in the absence (upper) and presence (lower) of 10 μM GAL-021. (B) Averaged I-V relationships of IK(DR) obtained in the control (■) and during cell exposure to 10 μM GAL-021 (□). Current amplitude was measured at the end of each voltage step, and each data point represents the mean ± SEM (n = 7). Of note, GAL-021 at a concentration of 10 μM mildly suppresses IK(DR) amplitude.

Figure 8
Figure 8

Inability of GAL-021 to suppress erg-mediated K+ current (IK(erg)) obtained in GH3 cells. We bathed cells in high-K+, Ca2+-free solution and the pipette used was filled with K+-containing solution. (A) Superimposed IK(erg) traces in the control (upper) and during the exposure to 10 μM GAL-021 (lower). The IK(erg) was activated by a series of long-step voltage pulse indicated in the uppermost part of (A). (B) Averaged I-V relationship of deactivating IK(erg) in the absence and presence of GAL-021 (10 μM) or GAL-021 (10 μM) plus telmisartan (Tel, 10 μM). Current amplitude was measured at the beginning of each hyperpolarizing voltage step. Each data point represents the mean ± SEM (n = 7 to 8). Note that GAL at a concentration of 10 μM was unable to suppress IK(erg) in these cells.

Figure 9
Figure 9

Inhibitory effect of GAL-021 on hyperpolarization-activated cationic current (Ih) identified in GH3 cells. In these whole-cell current recordings, cells were bathed in Ca2+-free, Tyrode’s solution, and the recording pipette was filled with K+-containing solution. (A) Representative Ih traces obtained in the absence (upper) or presence (lower) of 30 μM GAL-021. The uppermost part indicates the voltage-clamp protocol applied. (B) Averaged I-V relationships of Ih amplitude obtained in the control (i.e., in the absence of GAL-021), during the exposure to 30 μM GAL-021 and washout of GAL-021 (mean ± SEM and n = 8 for each data point). Current amplitude was collected at the end of 2 sec hyperpolarizing potential.

Figure 9
Figure 9

Inhibitory effect of GAL-021 on hyperpolarization-activated cationic current (Ih) identified in GH3 cells. In these whole-cell current recordings, cells were bathed in Ca2+-free, Tyrode’s solution, and the recording pipette was filled with K+-containing solution. (A) Representative Ih traces obtained in the absence (upper) or presence (lower) of 30 μM GAL-021. The uppermost part indicates the voltage-clamp protocol applied. (B) Averaged I-V relationships of Ih amplitude obtained in the control (i.e., in the absence of GAL-021), during the exposure to 30 μM GAL-021 and washout of GAL-021 (mean ± SEM and n = 8 for each data point). Current amplitude was collected at the end of 2 sec hyperpolarizing potential.

Figure 10
Figure 10

Effect of GAL-021 on BKCa-channel activity in α-hSlo-expressing HEK293T cells. Cells were suspended in high-K+ solution containing 0.1 μM Ca2+, and inside-out current recordings were made at the holding potential of +60 mV. (A) Representative BKCa-channel currents obtained in the control (left) and after bath application of 30 μM GAL-021 (right). (B) Summary bar graph depicting effect of GAL-021 (10 or 30 μM) and GAL-021 (30 μM) plus BMS-191011 (10 μM) on the open-state probability of BKCa (or KCNMA1) channels (mean ± SEM and n = 6 to 8 for each bar). *, Significantly different from control or GAL-021 (10 μM) alone group (p < 0.05); **, significantly different from control (p < 0.01); and †, significantly different from GAL-021 (30 μM) alone group (p < 0.05).

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