Figure 5. KCNN3 expression and KCa2.3 current inhibition by NS8593 in microglia in differing activation states.

A. Expression of KCNN3 mRNA was quantified using NanoString nCounter analysis in unstimulated primary microglia, and at 6 hr (solid bars) and 24 hr (striped bars) after treatment with 20 ng/ml IL4- or IL10-treatment. B–D. NS8593 inhibits the KCa2.3 current, while AA-861 has no effect. KCa2.3 currents were recorded from MLS-9 microglial cells in the perforated-patch configuration produced by amphotericin B (200 µg/ml) in the pipette. The voltage protocol throughout was a 120 ms-long voltage ramp from −100 to +80 mV from a holding potential of −70 mV. The bath always contained 1 µM TRAM-34 to block KCa3.1 currents. Riluzole (300 µM) was used simply as a tool to activate the KCa2.3 current [21]. B. Upper panel: Representative traces show the current evoked by riluzole with or without 7 µM NS8593. Lower panel: The representative time course (current measured at +80 mV) illustrates KCa2.3 current activation and its inhibition by NS8593. C. Representative currents and time course show that no current was activated when 7 µM NS8593 was present in the bath. D. The current is insensitive to 10 µM AA-861. Note that current activation by riluzole is readily reversible (wash), as we previously showed [21]. E–G. The KCa2.3 current in primary rat microglial cells is inhibited by 7 µM NS8593 under differing activation states. Currents were recorded using the same patch-clamp configuration as described for MLS-9 cells. Upper panels: Representative currents in microglia that were unstimulated (E), or treated for 24 hr with 20 ng/mL IL4 (F) or 20 ng/mL IL10 (G). Lower panels: A representative time course for each cell (current at +80 mV) shows activation by riluzole and inhibition by NS8593.