Key points A computational model of P2X channel activation in microglia was developed that includes downfield Ca2+‐dependent signalling pathways. This model provides quantitative insights into how diverse signalling pathways in microglia converge to control microglial function. Abstract Microglia function is orchestrated through highly coupled signalling pathways that depend on calcium (Ca2+). In response to extracellular ATP, transient increases in intracellular Ca2+ driven through the activation of purinergic receptors, P2X and P2Y, are sufficient to promote cytokine synthesis. Although the steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic purinergic receptors, P2X4 and P2X7, with the graded production of a gene product, namely the tumour necrosis factor α (TNFα) cytokine. In addition to Ca2+ handling mechanisms common to eukaryotic cells, our model includes microglia‐specific processes including ATP‐dependent P2X4 and P2X7 activation, activation of nuclear factor of activated T‐cells (NFAT) transcription factors, and TNFα production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNFα production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNFα production. Our results suggest that pulsatile stimulation of P2X4 via micromolar ATP may be sufficient to promote TNFα production, whereas high‐amplitude ATP exposure is necessary for production via P2X7. Furthermore, under conditions that increase P2X4 expression, for instance, following activation by pathogen‐associated molecular factors, P2X4‐associated TNFα production is greatly enhanced. Given that Ca2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology.

Calcineurin (CaN) is a calcium-dependent phosphatase involved in numerous signaling pathways. Its activation is in part driven by the binding of calmodulin (CaM) to a CaM recognition region (CaMBR) within CaN’s regulatory domain (RD). However, secondary interactions between CaM and the CaN RD may be necessary to fully activate CaN. Specifically, it is established that the CaN RD folds upon CaM binding and a region C-terminal to CaMBR, the “distal helix”, assumes an α-helix fold and contributes to activation [Dunlap, T. B., et al. (2013) Biochemistry 52, 8643–8651]. We hypothesized in that previous study that this distal helix can bind CaM in a region distinct from the canonical CaMBR. To test this hypothesis, we utilized molecular simulations, including replica-exchange molecular dynamics, protein–protein docking, and computational mutagenesis, to determine potential distal helix-binding sites on CaM’s surface. We isolated a potential binding site on CaM (site D) that facilitates moderate-affinity interprotein interactions and predicted that mutation of site D residues K30 and G40 on CaM would weaken CaN distal helix binding. We experimentally confirmed that two variants (K30E and G40D) indicate weaker binding of a phosphate substrate p-nitrophenyl phosphate to the CaN catalytic site by a phosphatase assay. This weakened substrate affinity is consistent with competitive binding of the CaN autoinhibition domain to the catalytic site, which we suggest is due to the weakened distal helix–CaM interactions. This study therefore suggests a novel mechanism for CaM regulation of CaN that may extend to other CaM targets.

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Membrane lipid composition is a critical feature of cell function, where cholesterol is a major lipid sterol component that influences the membranes physical and electrical properties. The effects of cholesterol on transport properties between adjacent to the cells, especially in junctions formed between cells is not completely understood. These junctions where substances transport and signaling is critical may be affected by modifying the cholesterol composition of the membrane in these junctional regions. Here we show how the cholesterol content in a membrane can regulate these phenomena by changing their effect on transport into and through regions between cell membranes in close proximity. Through geometric and electrostatic effects interaction with substrates, the properties of the fluid between membranes are shown to potentially enforce concentration gradients of dissolved compounds that may be biologically significant.

Microglia function is orchestrated through highly-coupled signaling pathways that depend on calcium (Ca 2+ ). In response to extracellular adenosine triphosphate (ATP), transient increases in intracellular Ca 2+ driven through the activation of purinergic receptors, P 2 X and P 2 Y, are sufficient to promote cytokine synthesis and potentially their release. While steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic purinergic receptors, P 2 X 4 and P 2 X 7 , with the graded production of a gene product, namely the tumor necrosis factor a (TNFa) cytokine. In addition to Ca 2+ handling mechanisms common to eukaryotic cells, our model includes microglia-specific processes including ATP-dependent P 2 X 4 and P 2 X 7 activation, activation of NFAT transcription factors, and TNFa production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNFa production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNFa production. Our key findings are that TNFa production via P 2 X 4 is maximized at low ATP when subject to high frequency ATP stimulation, whereas P 2 X 7 contributes most significantly at millimolar ATPranges. Given that Ca 2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology.