Pioglitazone, a PPAR-γ Activator, Stimulates BKCa but Suppresses IK M in Hippocampal Neurons

Abstract

Pioglitazone (PIO), a thiazolidinedone, was reported to stimulate peroxisome proliferator-activated receptor-γ (PPAR-γ) with anti-inflammatory, anti-proliferative, anti-diabetic, and antidepressive activities. However, whether this compound exerts any perturbations on Ca²⁺-activated K⁺ and M-type K⁺ currents in central neurons remains largely unresolved. In this study, we investigated the effects of PIO on these potassium currents in hippocampal neurons (mHippoE-14). In whole-cell current recordings, the presence of PIO (10 μM) increased the amplitude of Ca²⁺-activated K⁺ current [I_K(Ca)] in mHippoE-14 cells. PIO-induced stimulation of I_K(Ca) observed in these cells was reversed by subsequent addition of paxilline, yet not by TRAM-39 or apamin. In inside-out current recordings, PIO applied to the bath concentration-dependently increased the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with an EC₅₀ value of 7.6 μM. Its activation of BK_Ca channels in mHippoE-14 cells was voltage-dependent and accompanied by both a lengthening in mean open time and a shortening in slow component of mean closed time. The activation curve of BK_Ca channels after addition of PIO was shifted to less depolarized potential without any change in the gating charge. PIO also suppressed the amplitude of M-type K⁺ currents inherently in mHippoE-14 neurons. Taken together, in addition to its agonistic action on PPAR-γ, PIO-induced perturbation of these potassium channels may be responsible for its widely pharmacological actions on hippocampal neurons.

Keywords: Ca2+-activated K+ current; M-type K+ current; hippocampal neuron; large-conductance Ca2+-activated K+ channel; pioglitazone.

FIGURE 1

Effect of PIO on whole-cell Ca²⁺-activated K⁺ current (I_K(Ca)) in mHippoE-14 hippocampal neurons. In these experiments, cells were bathed in normal Tyrode’s solution, the composition of which was described under Section “Materials and Methods.” The recording pipette was filled with K⁺-containing solution. (A) Superimposed I_K(Ca) traces obtained in the control (middle part) and during cell exposure to 10 μM PIO (bottom part). The upper part indicates the voltage protocol applied, and arrowheads are zero current level. (B) Averaged I–V relationships of I_K(Ca) obtained in the control (), during the exposure () to 10 μM PIO and after washout () of PIO (mean ± SEM; n = 11 for each point). ^∗Significantly different from control groups taken at the same level of voltage pulse. (C) Bar graph showing summary of the effect of PIO, PIO plus TRAM-39, PIO plus apamin, and PIO plus paxilline, and PIO plus tolbutamide on I_K(Ca) amplitude (mean ± SEM; n = 10–12 for each bar). Current amplitude was measured at +50 mV. (a) Control; (b) 10 μM PIO; (c) 10 μM PIO plus 3 μM TRAM-39; (d) 10 μM PIO plus 200 nM apamin; (e) 10 μM PIO plus 1 μM paxilline; (f) 10 μM PIO plus 30 μM tolbutamide. ^∗Significantly different from control (P < 0.05) and ^∗∗significantly different from PIO alone group (P < 0.05) (n = 9–10 for each bar).

FIGURE 2

Effect of PIO on BK_Ca channel activity in mHippoE-14 hippocampal neurons. (A) Original current traces of BK_Ca channels obtained in the absence (left) and presence (right) of 10 μM pioglitazone (PIO). The examined cells were bathed in symmetrical K⁺ solution (145 mM). Under inside-out current recordings, the potential was held at +60 mV and bath medium contained 0.1 μM Ca²⁺. The upward deflection represents the opening event of the channel. The lower part indicates the expanded trace recorded from the uppermost part in the control and during exposure to PIO. (B) BK_Ca-channel trace obtained after washout of PIO. (C) Concentration-dependent increase in channel open probability (mean ± SEM; n = 9–11 for each point). Channel activity measured at +60 mV during the exposure to 100 μM PIO was taken to be 100%. The values for EC₅₀, Hill coefficient and maximal percentage increase of BK_Ca channels in the presence of PIO were 7.6 μM 1.3 and 100%, respectively.

FIGURE 3

Mean-variance histogram of BK_Ca channels taken from the absence (A) and presence (B) of 10 μM PIO. The examined cells were bathed in symmetrical K⁺ solution, bath medium contained 0.1 μM Ca²⁺ and the holding potential was set at +60 mV. The closed state corresponds to the peak at 0 pA. The mean currents (indicated by asterisk) in (A,B) were 10.9 and 10.7 pA, respectively.

FIGURE 4

Effect of PIO on mean open- (A) and closed-time (B) histograms of BK_Ca channels recorded from mHippoE-14 hippocampal neurons. The holding potential was set at +60 mV, and inside-out configuration was performed. In control (left side), the open-time histogram of the channel was fitted by a single exponential function (indicated by red smooth line) with a mean open time of 1.9 ms, while the closed-time histogram was by a sum of a two-exponential function with a mean closed time of 3.5 and 47.5 ms. After addition of 10 μM PIO (right side), the mean open time was increased to 2.7 ms, and the slow component of closed time was shortened to 28.7 ms; however, minimal change in the fast component of closed time (i.e., 3.4 ms) in the presence of this compound. Of note, the abscissa and ordinate in each histogram indicate the logarithm of open or closed time (ms) and the square root of even number, respectively. Data were taken from a measurement of 100 channel openings. The vertical black dashed lines are placed at the values of mean open or closed time for BK_Ca channels.

FIGURE 5

Effect of PIO on the I–V relation of BK_Ca channels in mHippoE-14 hippocampal neurons. The experiments on BK_Ca channels were conducted with symmetrical K⁺-rich concentration (145 mM). Under inside-out configuration, the potential was held at +60 mV and bath medium medium contained 0.1 μM Ca²⁺. (A) Original current traces obtained in the control and during exposure to 10 μM PIO. The labels in the rightmost side indicate the holding potential applied. Arrowhead in each trace corresponds to zero current level, and the upper deflection indicates the opening event of the channel. In (B), the single-channel conductance in the absence () and presence () of 10 μM PIO is nearly identical. Each point represents mean ± SEM (n = 9–10). The dashed red lines obtained with or without addition of PIO are pointed toward the values of the reversal potential (i.e., 0.0 ± 0.1 mV, n = 8). (C) The relationship between relative open probability of BK_Ca channels and membrane potential obtained with or without addition of 10 μM PIO. The ramp pulses were applied from 0 to +80 mV with a duration of 1 s. Under inside-out current recordings, PIO (10 μM) was applied to the intracellular surface of the excised patch. The smooth lines represent the best fit to the Boltzmann equation as detailed in Section “Materials and Methods.”

FIGURE 6

Effect of linopirdine and linopirdine plus flupirtine on the amplitude of M-type K⁺ current [I_K(M)] in mHippoE-14 hippocampal neurons. In this set of experiments, cells were bathed in high K⁺, Ca²⁺-free solution and the recording pipette was filled with K⁺-containing solution. (A) Superimposed I_K(M) traces obtained in the control (a) and during the exposure to 10 μM linopirdine (b), and 10 μM linopirdine plus 10 μM flupirtine (c). The upper part indicates the voltage protocol used. (B) Bar graph showing the effect of linopirdine and linopirdine plus flupirtine on I_K(M) amplitude (mean ± SEM; n = 9 for each bar). ^∗Significantly different from control (P < 0.05). LINO, linopirdine; FLUP, flupirtine.

FIGURE 7

Effect of PIO on I_K(M) amplitude in mHippoE-14 hippocampal neurons. These experiments were conducted in cells bathed in high K⁺, Ca²⁺-free solution and the recording pipette was filled with K⁺-containing solution. (A) Superimposed I_K(M) traces obtained in the absence (a) and presence of 3 μM (b), and 10 μM PIO (c). The upper part indicates the voltage protocol used. (B) Bar graph showing the effect of PIO, linopirdine, and PIO plus flupirtine on I_K(M) amplitude (mean ± SEM; n = 9–11 for each bar). The I_K(M) amplitude elicited by membrane depolarization from –50 to –10 mV was measured. (a) Control; (b) 10 μM PIO; (c) 10 μM linopirdine; (d) 10 μM PIO plus 10 μM flupirtine. ^∗Significantly different from control (P < 0.05) and ^∗∗significantly different from PIO (10 μM) alone group (P < 0.05). LINO, linopirdine; FLUP, flupirtine.