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  • Prostaglandin J2 br The statistical analysis was

    2022-04-26


    The statistical analysis was carried out by using SPSS statistics 22.0 software (IBM Corporation, Armonk, NY, USA). Data are presented as the mean ± SEM. For electrophysiological experiments, statistical comparisons were made by unpaired or one-sample Student's t-test. For cell migration, the difference between groups were compared by one-way ANOVA followed by post hoc Dunnett's test. Statistical significance was set at p < 0.05.
    3. Results
    The effects of chloroquine were first examined using the whole-cell configuration of the patch clamp technique. Kv10.1 currents were eli-cited by 500-ms depolarizing steps to +60 mV, followed by repolar-ization to −70 mV. The voltage protocol was repeated every 10-s. Kv10.1 channels were inhibited by chloroquine in a concentration-de-pendent manner (Fig. 1). Fig. 1A shows a temporal course of Kv10.1 currents in response to external application of 30 μM chloroquine.
    Fig. 1. Effect of chloroquine (CQ) on Kv10.1 channels expressed in HEK293 Prostaglandin J2 and re-corded in whole-cell configuration. (A) Representative time course of the effect of 30 μM chloroquine on Kv10.1 currents. (B) Representative Kv10.1 current traces in absence (control) and presence of chloroquine at in-creasing concentrations (3–300 μM). (C) Concentration-response relationship for Kv10.1 current inhibition by chloroquine at +60 mV. Mean values were plotted against chloroquine concentration and fitted with the Hill equation. Mean IC50 was 31.05 ± 4.5 μM and the Hill coefficient, nH = 0.88 ± 0.1 (n = 7).
    Fig. 2. Block of Kv10.1 currents by chloroquine (CQ) is voltage-dependent. (A) Representative Kv10.1current traces obtained with the protocol shown below panel B. (B) Currents recorded in presence of 30 μM chloroquine in the same cell depicted in panel A. inset, superimposed tail currents obtained at −50 mV after a 500-ms depolarization to +80 mV. (C) Normalized current-voltage re-lationship for currents measures at the end of the 500-ms pulses in control and in the presence of chloroquine (n = 9). (D) Fractional block (1 – Idrug/Icontrol) of Kv10.1 currents plotted as a function of test potential (n = 9). Idrug and Icontrol were measured at the end of the depolarizing pulse at each membrane poten-tial.
    We further explored the effect of chloroquine on Kv10.1 by re-cording currents in a voltage range from −60 to +80 mV, applied in 10-mV increments from a holding potential of −80 mV. In the absence of drug, Kv10.1 currents slowly activate and does not inactivate (Fig. 2A). In the presence of drug (30 μM chloroquine), the currents are profoundly inhibited and showed a crossover at potentials above +50 mV (Fig. 2B). Plots of the maximum current amplitudes measured at the end of the 500-ms pulses indicate that block by chloroquine (30 μM) was voltage-dependent with more pronounced reductions at more depolarized potentials (Fig. 2C–D).
    The inset in Fig. 2A shows the superposition of the tail currents obtained at −50 mV after a 500-ms depolarization to +80 mV. The tail current declined slower in the presence of chloroquine compared to that of control, resulting in Prostaglandin J2 the crossover phenomenon.
    To determine whether chloroquine acts from the extracellular or intracellular side of the plasma membrane, inside-out current record-ings were performed. Under this configuration, chloroquine inhibited Kv10.1 currents with a faster temporal course (Fig. 3A); the time to reach the steady-state current inhibition was ∼20-s when it was ap-plied to the intracellular side, compared to ∼200-s of the extracellular application. Fig. 3B shows current traces obtained under control con-ditions and during perfusion of the cell with chloroquine at different concentrations (3, 10, 30, 100 and 300 μM). The concentration-re-sponse relationship shows that the IC50 was 31.72 ± 4.2 μM and the Hill coefficient, nH = 0.85 ± 0.08 (Fig. 3C), which was not sig-nificantly different from that obtained under whole-cell configuration. Next, we added 30 μM chloroquine to the inside-out pipette solution to restrict its action to the external side of the plasma membrane while 
    maintaining a constant bath solution perfusion to avoid the accumu-lation of chloroquine that diffused across the plasma membrane. Under this conditions, chloroquine did not inhibited Kv10.1 currents; how-ever, currents were quickly inhibited when chloroquine was directly applied to the internal bath solution (Fig. 4A). Fig. 4B shows re-presentative current traces obtained at the points indicated in the figure (4A, P1-P3). Finally, we applied chloroquine to the internal side of the plasma membrane during a long depolarization. Chloroquine (30 μM) was applied for 5-s during a 20-s depolarizing pulse to +60 mV (Fig. 4C); after the current was completely activated, the application of chloroquine induced a fast inhibition that was fully recovered with the perfusion of drug-free solution. The results above strongly support the idea that chloroquine inhibits Kv10.1 channels from the cytoplasmic side of the plasma membrane.