Differential contribution of THIK-1 K+ channels and P2X7 receptors to ATP-mediated neuroinflammation by human microglia

Abstract

Neuroinflammation is highly influenced by microglia, particularly through activation of the NLRP3 inflammasome and subsequent release of IL-1β. Extracellular ATP is a strong activator of NLRP3 by inducing K⁺ efflux as a key signaling event, suggesting that K⁺-permeable ion channels could have high therapeutic potential. In microglia, these include ATP-gated THIK-1 K⁺ channels and P2X7 receptors, but their interactions and potential therapeutic role in the human brain are unknown. Using a novel specific inhibitor of THIK-1 in combination with patch-clamp electrophysiology in slices of human neocortex, we found that THIK-1 generated the main tonic K⁺ conductance in microglia that sets the resting membrane potential. Extracellular ATP stimulated K⁺ efflux in a concentration-dependent manner only via P2X7 and metabotropic potentiation of THIK-1. We further demonstrated that activation of P2X7 was mandatory for ATP-evoked IL-1β release, which was strongly suppressed by blocking THIK-1. Surprisingly, THIK-1 contributed only marginally to the total K⁺ conductance in the presence of ATP, which was dominated by P2X7. This suggests a previously unknown, K⁺-independent mechanism of THIK-1 for NLRP3 activation. Nuclear sequencing revealed almost selective expression of THIK-1 in human brain microglia, while P2X7 had a much broader expression. Thus, inhibition of THIK-1 could be an effective and, in contrast to P2X7, microglia-specific therapeutic strategy to contain neuroinflammation.

Keywords: Human brain; Ion channels; Microglia; Neocortex; Neuroinflammation; Pharmacology; Purinergic signalling.

Fig. 1

THIK-1 regulates the electrical membrane properties in microglia in the human brain. A Top: anatomical localisation of human temporal lobe from which acute brain slices were prepared. Bottom: specimen images of morphologically identified microglia patch clamped in human (top) and murine (bottom) brain slices (scale bar, 50 µm) and after filling them with Alexa-488 via the patch solution (scale bar, 20 µm). B Membrane currents with capacitive and leak components in response to 10 mV hyperpolarizing voltage steps of human und murine microglia, from which electrical membrane properties were determined. C, D Comparison of input resistance (C), and resting membrane potential (D) of human and murine microglia. E Specimen current profiles in response to voltage steps from − 150 to + 60 mV of a human and murine microglia (left) and respective plot of voltage dependence for all cells per condition (right). F Effect of THIK-1 inhibitor, C100814, on the resting membrane potential of human microglia, illustrating the time course of changes of an individual cell (left) and the resulting mean depolarization (right). G Specimen recording showing C100814-induced changes in tonic THIK-1 current (left) and voltage dependence of respective C100814-inhibited current (right), revealing a reversal potential of ~ − 90 mV close to the predicted K⁺ equilibrium potential (inset, right). H Time course of changes in resting membrane potential by C100814 in murine microglia (left) and quantification of effects in comparison to mice lacking THIK-1 (THIK-1 KO) (right). Note the similar degree of depolarization obtained after pharmacological blockade and genetic deletion. I Example trace showing the lack of effect of C100814 on baseline current in microglia from THIK-1 KO mice (left) and quantification of effect relative to values before drug application (right). Data information: data indicate mean ± SEM. Dots on bars show number of cells. Data are from four individual patients, and four mice. P-values are from unpaired (C, D, H) and paired (F, H, I) Student’s t tests

Fig. 2

Extracellular ATP increases K⁺ efflux from human microglia via THIK-1 and P2X7 receptors in a concentration-dependent manner. A Schematic illustrating the potentiation of THIK-1 K⁺ current via metabotropic coupling to high-affinity P2Y12 receptors, which transiently hyperpolarizes microglia to ~ − 60 mV. B Specimen outward THIK-1 current elicited by 100 µM ATP (left) showing rapid desensitization in the continued presence of ATP, analysed as residual current present 5 min after peak response (right). Mean current amplitude of ATP-evoked THIK-1 current was 19.25 pA ± 3.7 (n = 9). C Current profiles of a single microglia to voltage steps from − 150 mV to + 60 mV before and during application of 100 µM ATP (left) and respective voltage dependence of the net ATP-evoked THIK-1 current after subtracting (2) − (1) (right). Inset indicates the reversal potential of ~ − 85 mV, close to the predicted equilibrium potential for K⁺. D Effect of 5 µM C100814 on repeatedly evoked THIK-1 current by brief pulses of locally applied 100 µM ATP (left). Note suppression of the ATP-evoked and tonic THIK-1 current components (shaded area). Quantification of C100814-induced inhibition of ATP-evoked THIK-1 current, normalised to the mean outward current prior to drug application in the same cell (right). E Schematic showing ion channel pathways activated in microglia by exposure to high extracellular ATP concentration (> 1 mM). Additional activation of P2X7 cation channels, depolarizes microglia to ~ 0 mV, which concomitantly enhances K⁺ efflux via tonically active THIK-1 channels (dashed arrow) due to an increased driving force for K⁺. F Time course of microglial membrane current to 5 mM ATP at a holding potential of − 30 mV showing the emergence of a large sustained inward current that does not desensitize in the continued presence of ATP (left). Quantification of the ATP-evoked inward current 7 min after reaching steady state (2), normalised to current at time (1) when steady state was initially reached (right). Mean current amplitude of ATP-evoked P2X7 was − 103.42 pA ± 59.6 (n = 5). G Current profile of a single microglia to voltage steps from − 150 to + 60 mV before and during application of 5 mM ATP (left) and respective voltage-dependence of the net ATP-evoked P2X7 current after subtracting (2) − (1) (right). Inset indicates the reversal potential of ~ + 5 mV, close to the predicted equilibrium potential of a nonselective cation channel (P2X7). H Effect of 20 µM P2X7 antagonist A740003 on repeatedly evoked P2X7 current by brief pulses of locally applied 5 mM ATP at a holding potential of − 30 mV (left). Quantification of A74003-mediated inhibition of ATP-evoked P2X7 current, normalised to the mean inward current before drug application in the same cell (right). Data information: data indicate mean ± SEM. Dots on bars show number of cells. Data are from four individual patients. P-values are from paired Student’s t tests

Fig. 3

Blockade of THIK-1 suppresses ATP-evoked IL-1β release from microglia in human brain. A Comparison of expression levels (mRNA) of P2X7, THIK-1, ASC, NLRP3, Caspase 1 and IL-1β for major CNS cell types analysed by nuclear enriched transcript sort sequencing (NETSseq) of healthy human brain tissue donors (n = 32). B Workflow illustrating determination of IL-1β levels upon acute activation of microglia in human and murine brain slices by purinergic stimulation (5 mM ATP, 3 h) as a model to simulate acute brain pathology. C–E Effects on IL-1β release in response to THIK-1 activation by 100 µM ATP (C) or in addition by P2X7 activation with 5 mM ATP for 3 individual human donors (D), and in comparison to mouse brain slices (E). Note the requirement for high ATP (i.e., P2X7 activation) and the dispensability of a priming stimulus to trigger NLRP3-dependent IL-1β release in both human and mouse brain that is abolished by the NLRP3 antagonist MCC950. Data are normalized to control condition and are from four individual patients and four wild-type mice. P values refer to 5 mM ATP condition. F, G Suppression of IL-1β release triggered by 5 mM ATP upon blockade of THIK-1 and P2X7 in human (F) and murine brain slices (G). Data are normalised to 5 mM ATP condition and are from three individual patients or four wildtype mice. P-values refer to 5 mM ATP condition. Data information: data indicate mean ± SEM. Dots on bars show number of slices. P-values are from unpaired Student’s t tests

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 V_m = 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