Fig. 4
THIK-1, as opposed to P2X7, contributes only marginally to K+ efflux for ATP-triggered NLRP3 activation. A Confocal images showing GFP-encoded microglia (green) in cortical murine slices before (control) and after microglial activation by ATP (5 mM, 3 h). Scale bar, 10 µm. B Exemplary voltage clamp recording of murine microglia at a holding potential of 0 mV (i.e., the approximate reversal potential of P2X7), under similar experimental conditions as those used to study IL-1β release. Schematics depict the stepwise pharmacological isolation of THIK-1 and P2X7 to analyse respective K+ conductance ratios from evoked current transients. C Current transients in response to 40 mV hyperpolarizing voltage steps of murine microglia (at Vm = 0 mV) used to determine THIK-1 conductance by subtracting (1) − (2) and P2X7 conductance by subtracting (3) − (2). Note the different scale on the far right to display the much larger P2X7-evoked current. D, E Quantification of the individual K+ conductance mediated by P2X7 and THIK-1 for nonactivated (D) and activated microglia (E), considering a K+ conductance of 100% for THIK-1 and 45% for P2X7 (see “Methods” for details). Obtained conductance ratios reflect the sustained efflux of K+ from microglia in the continued presence of ATP accomplished by nondesensitizing P2X7 and tonic THIK-1 currents (because the ATP-potentiated THIK-1 current is transient and rapidly decays with a tau of 2.5 min). F Analysis of the resting membrane potential of nonactivated (control) and activated microglia after purinergic stimulation. G Specimen current profiles of a nonactivated (left) and ATP-activated (middle) microglia to voltage steps from − 150 to + 60 mV at a holding potential of − 60 mV. Plot of current–voltage dependencies averaged for all cells per condition (right). Note the more positive reversal potential of activated compared to control microglia and the absence of upregulation of other voltage-gated ion channels after purinergic activation. Data information: data indicate mean ± SEM. Dots on bars show number of cells. Data are from three wildtype mice. P-values are from unpaired Student’s t tests