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Cited by 212Inflammasome and immune disordersAdenosine and Purinergic SignalingCalcium signaling and nucleotide metabolism

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K+ regulates Ca2+ to drive inflammasome signaling: dynamic visualization of ion flux in live cells

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Yaron, J R, Gangaraju, S, Rao, M Y, Kong, X, Zhang, L, Su, F, Tian, Y, Glenn, H L, Meldrum, D R·2015·Cell Death & Disease
DOI 10.1038/cddis.2015.277PMC PMC5399176
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Abstract

P2X7 purinergic receptor engagement with extracellular ATP induces transmembrane potassium and calcium flux resulting in assembly of the NLRP3 inflammasome in LPS-primed macrophages. The role of potassium and calcium in inflammasome regulation is not well understood, largely due to limitations in existing methods for interrogating potassium in real time. The use of KS6, a novel sensor for selective and sensitive dynamic visualization of intracellular potassium flux in live cells, multiplexed with the intracellular calcium sensor Fluo-4, revealed a coordinated relationship between potassium and calcium. Interestingly, the mitochondrial potassium pool was mobilized in a P2X7 signaling, and ATP dose-dependent manner, suggesting a role for mitochondrial sensing of cytosolic ion perturbation. Through treatment with extracellular potassium we found that potassium efflux was necessary to permit sustained calcium entry, but not transient calcium flux from intracellular stores. Further, intracellular calcium chelation with BAPTA-AM indicated that P2X7-induced potassium depletion was independent of calcium mobilization. This evidence suggests that both potassium efflux and calcium influx are necessary for mitochondrial reactive oxygen generation upstream of NLRP3 inflammasome assembly and pyroptotic cell death. We propose a model wherein potassium efflux is necessary for calcium influx, resulting in mitochondrial reactive oxygen generation to trigger the NLRP3 inflammasome.

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Figure 1 P2X7-induced potassium efflux regulates NLRP3 inflammasome assembly and pyroptotic cell death. (a) Immunoblot analysis of procaspase-1 p45 and activated p10 fragments, and proIL-1β (34 kD) and mature (17 kD) fragments in the lysates and concentrated supernatants of J774A.1 cells primed for 4 h with 1 μg/ml LPS and stimulated with 3 mM ATP for 30 min with or without addition of 130 mM extracellular KCl or 25 μM of the P2X7 antagonist A438079. (b) Propidium iodide in J774A.1 cells primed with LPS for 4 h and stimulated with ATP in the presence or absence of 130 mM extracellular KCl. (c) Caspase-1 FLICA staining (green) in J774A.1 cells untreated or primed for 4 h with 1 μg/ml LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 130 mM extracellular KCl or 25 μM A438079. Arrows: caspase-1 specks indicative of inflammasome assembly. Scale bar represents 50 μM. Nuclei are stained with NucBlue Fixed DAPI solution (blue). (d) Image cytometry analysis of inflammasomes detected by Caspase-1 FLICA. J774A.1 cells untreated or primed for 4 h with 1 μg/ml LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 130 mM extracellular KCl or 25 μM A438079. Bar graph represents mean counts and standard error from at least 4000 cells for each condition from at a minimum of three fields in two independent experiments. Statistics were calculated by one-way ANOVA with Tukey's post hoc analysis
Figure 1 P2X7-induced potassium efflux regulates NLRP3 inflammasome assembly and pyroptotic cell death. (a) Immunoblot analysis of procaspase-1 p45 and activated p10 fragments, and proIL-1β (34 kD) and mature (17 kD) fragments in the lysates and concentrated supernatants of J774A.1 cells primed for 4 h with 1 μg/ml LPS and stimulated with 3 mM ATP for 30 min with or without addition of 130 mM extracellular KCl or 25 μM of the P2X7 antagonist A438079. (b) Propidium iodide in J774A.1 cells primed with LPS for 4 h and stimulated with ATP in the presence or absence of 130 mM extracellular KCl. (c) Caspase-1 FLICA staining (green) in J774A.1 cells untreated or primed for 4 h with 1 μg/ml LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 130 mM extracellular KCl or 25 μM A438079. Arrows: caspase-1 specks indicative of inflammasome assembly. Scale bar represents 50 μM. Nuclei are stained with NucBlue Fixed DAPI solution (blue). (d) Image cytometry analysis of inflammasomes detected by Caspase-1 FLICA. J774A.1 cells untreated or primed for 4 h with 1 μg/ml LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 130 mM extracellular KCl or 25 μM A438079. Bar graph represents mean counts and standard error from at least 4000 cells for each condition from at a minimum of three fields in two independent experiments. Statistics were calculated by one-way ANOVA with Tukey's post hoc analysisCC BY · PubMed Central
Figure 2 Calcium influx is an upstream regulator of IL-1β release and NLRP3 inflammasome assembly. (a) ELISA analysis of released IL-1β from J774A.1 cells primed with 1 μg/ml LPS for 4 h and stimulated with 3 mM ATP for 30 min. Where indicated, cells were pretreated with 100 μM BAPTA-AM before addition of ATP. Statistics were calculated by one-way ANOVA with Tukey's post hoc and represent the mean and standard error of two independent experiments. (b) Cells were prepared as in (a), except for the addition of caspase-1 FLICA 1 h before the addition of ATP (green). Arrows indicate perinuclear caspase-1 specks. Blue fluorescence indicates nuclei stained with NucBlue Fixed DAPI solution. Scale bar represents 50 μM. (c) Image cytometry analysis of inflammasomes detected by caspase-1 FLICA. Bar graph represents mean counts and standard error from at least 4000 cells for each condition from at a minimum of three fields in two independent experiments. Statistics were calculated by one-way ANOVA with Tukey's post hoc analysis
Figure 2 Calcium influx is an upstream regulator of IL-1β release and NLRP3 inflammasome assembly. (a) ELISA analysis of released IL-1β from J774A.1 cells primed with 1 μg/ml LPS for 4 h and stimulated with 3 mM ATP for 30 min. Where indicated, cells were pretreated with 100 μM BAPTA-AM before addition of ATP. Statistics were calculated by one-way ANOVA with Tukey's post hoc and represent the mean and standard error of two independent experiments. (b) Cells were prepared as in (a), except for the addition of caspase-1 FLICA 1 h before the addition of ATP (green). Arrows indicate perinuclear caspase-1 specks. Blue fluorescence indicates nuclei stained with NucBlue Fixed DAPI solution. Scale bar represents 50 μM. (c) Image cytometry analysis of inflammasomes detected by caspase-1 FLICA. Bar graph represents mean counts and standard error from at least 4000 cells for each condition from at a minimum of three fields in two independent experiments. Statistics were calculated by one-way ANOVA with Tukey's post hoc analysisCC BY · PubMed Central
Figure 3 KS6 localizes to predominantly mitochondria in live cells. (a) Chemical structure of the intracellular potassium sensor KS6. (b) Spectrofluorophotometric characterization of KS6 sensor signal response to potassium titration in solution. (c) J774A.1 cells were stained with KS6 intracellular potassium sensor and MitoTracker Green FM before imaging by confocal microscopy. (d) Inset of boxed region from (c) displaying the overlap of MitoTracker Green FM and KS6. (e) Signal from MitoTracker Green FM. (f) Signal from KS6. Arrows indicate discrete mitochondria clearly stained for both probes. Scale bar represents 20 μM in (c) and 10 μM in (d)
Figure 3 KS6 localizes to predominantly mitochondria in live cells. (a) Chemical structure of the intracellular potassium sensor KS6. (b) Spectrofluorophotometric characterization of KS6 sensor signal response to potassium titration in solution. (c) J774A.1 cells were stained with KS6 intracellular potassium sensor and MitoTracker Green FM before imaging by confocal microscopy. (d) Inset of boxed region from (c) displaying the overlap of MitoTracker Green FM and KS6. (e) Signal from MitoTracker Green FM. (f) Signal from KS6. Arrows indicate discrete mitochondria clearly stained for both probes. Scale bar represents 20 μM in (c) and 10 μM in (d)CC BY · PubMed Central
Figure 4 Real-time P2X7-dependent intracellular potassium dynamics observed with KS6. (a) Kinetic trace of potassium efflux from J774A.1 cells stimulated with 5 mM ATP at the indicated time point in the presence of 0 mM additional KCl (normal DMEM medium), 50 or 130 mM additional extracellular KCl. Traces represent the mean and standard error of 10–20 cells in each field. (b) Response at 40 min of potassium efflux (top panel) or TO-PRO-3 uptake (bottom panel) of J774A.1 cells primed for 4 h with 1 μg/ml LPS and treated with 1, 3 or 5 mM extracellular ATP. Bars represent mean and standard deviation of 20 cells in each condition. Statistics were performed by one-way ANOVA with Fischer's LSD comparison test. *Indicates P<0.05 and **** indicates P<0.0001. (c) Representative fields at the indicated time points of LPS-primed J774A.1 loaded with KS6 (red) and treated with 1, 3 or 5 mM extracellular ATP in the presence of TO-PRO-3 (cyan). Scale bar represents 50 μM. Pre-treatment of LPS-primed, ATP-stimulated J774A.1 macrophages with the P2X7 inhibitor A438079 suppresses (d) potassium efflux (e) and membrane permeability. Traces represent mean and standard error for five representative cells. Results are representative of at least two experiments
Figure 4 Real-time P2X7-dependent intracellular potassium dynamics observed with KS6. (a) Kinetic trace of potassium efflux from J774A.1 cells stimulated with 5 mM ATP at the indicated time point in the presence of 0 mM additional KCl (normal DMEM medium), 50 or 130 mM additional extracellular KCl. Traces represent the mean and standard error of 10–20 cells in each field. (b) Response at 40 min of potassium efflux (top panel) or TO-PRO-3 uptake (bottom panel) of J774A.1 cells primed for 4 h with 1 μg/ml LPS and treated with 1, 3 or 5 mM extracellular ATP. Bars represent mean and standard deviation of 20 cells in each condition. Statistics were performed by one-way ANOVA with Fischer's LSD comparison test. *Indicates P<0.05 and **** indicates P<0.0001. (c) Representative fields at the indicated time points of LPS-primed J774A.1 loaded with KS6 (red) and treated with 1, 3 or 5 mM extracellular ATP in the presence of TO-PRO-3 (cyan). Scale bar represents 50 μM. Pre-treatment of LPS-primed, ATP-stimulated J774A.1 macrophages with the P2X7 inhibitor A438079 suppresses (d) potassium efflux (e) and membrane permeability. Traces represent mean and standard error for five representative cells. Results are representative of at least two experimentsCC BY · PubMed Central
Figure 5 P2X7 activation results in mitochondrial potassium mobilization. J774A.1 cells were primed for 4 h with 1 μg/ml LPS and loaded with 5 μM KS6 (red) and 10 nM MitoTracker Green FM (green). Real-time confocal microscopy was performed to track the potassium dynamics after stimulation with 3 mM extracellular ATP with or without the P2X7 inhibitor A438079. Results revealed a rapid, receptor-dependent mobilization of potassium as indicated by a reduction in KS6 signal in the co-localized space with MitoTracker Green FM that was sensitive to inhibition with A438079. Subsequent to the mobilization, mitochondria appeared to fragment. Fields are representative of at least 3–5 experiments. Scale bar represents 20 μM
Figure 5 P2X7 activation results in mitochondrial potassium mobilization. J774A.1 cells were primed for 4 h with 1 μg/ml LPS and loaded with 5 μM KS6 (red) and 10 nM MitoTracker Green FM (green). Real-time confocal microscopy was performed to track the potassium dynamics after stimulation with 3 mM extracellular ATP with or without the P2X7 inhibitor A438079. Results revealed a rapid, receptor-dependent mobilization of potassium as indicated by a reduction in KS6 signal in the co-localized space with MitoTracker Green FM that was sensitive to inhibition with A438079. Subsequent to the mobilization, mitochondria appeared to fragment. Fields are representative of at least 3–5 experiments. Scale bar represents 20 μMCC BY · PubMed Central
Figure 6 Mitochondrial ROS is essential for ATP-evoked inflammasome activity and is driven by P2X7-dependent cation flux in J774A.1 cells. (a) Caspase-1 FLICA staining (green) in J774A.1 cells primed for 4 h with 1 μg/ml LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 500 μM MitoTEMPO treatment. Arrows point to caspase-1 specks indicative of inflammasome assembly. Scale bar represents 50 μM. Nuclei are stained with NucBlue Fixed DAPI solution (blue). (b) Image cytometry analysis of inflammasomes detected by caspase-1 FLICA. Bar graph represents mean counts and standard error from at least 4000 cells for each condition from at a minimum of three fields in two independent experiments. Statistics were calculated by one-way ANOVA with Tukey's post hoc analysis. (c) Immunoblot analysis of pro-caspase-1 p45 and activated p10 fragments, and proIL-1β (34 kD) and mature (17 kD) fragments in the lysates and concentrated supernatants of J774A.1 cells primed for 4 h with 1 μg/ml LPS and stimulated with 3 mM ATP for 30 min with or without pretreatment with 500 μM MitoTEMPO. (d) Assessment of lactate dehydrogenase (LDH) activity in the supernatants of J774A.1 cells primed with 1 μg/ml LPS and stimulated with ATP for 30 min with or without pretreatment with 500 μM MitoTEMPO. Results are fold-change versus LPS primed cells and error bars represent standard error of two independent experiments. ** Indicates P<0.01 by one-way ANOVA with Tukey's post hoc comparison. (e and f) J774A.1 cells were left untreated, primed with LPS for 4 h, or primed with LPS and treated with 3 mM ATP as indicated. MitoSOX (red) was added to all samples 15 min after ATP addition and incubated for an additional 15 min before live-cell imaging. (e) Evaluation of the role of P2X7 and potassium efflux in mROS generation. 130 mM KCl and 25 μM A438079 were added 15–20 min before ATP addition. (f) Evaluation of the role of calcium influx in mROS generation. 100 μM BAPTA-AM was added 15–20 min before ATP addition. Nuclei are stained with Hoechst 33342 (blue). Scale bar is 50 μm. Results are representative of at least two independent experiments
Figure 6 Mitochondrial ROS is essential for ATP-evoked inflammasome activity and is driven by P2X7-dependent cation flux in J774A.1 cells. (a) Caspase-1 FLICA staining (green) in J774A.1 cells primed for 4 h with 1 μg/ml LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 500 μM MitoTEMPO treatment. Arrows point to caspase-1 specks indicative of inflammasome assembly. Scale bar represents 50 μM. Nuclei are stained with NucBlue Fixed DAPI solution (blue). (b) Image cytometry analysis of inflammasomes detected by caspase-1 FLICA. Bar graph represents mean counts and standard error from at least 4000 cells for each condition from at a minimum of three fields in two independent experiments. Statistics were calculated by one-way ANOVA with Tukey's post hoc analysis. (c) Immunoblot analysis of pro-caspase-1 p45 and activated p10 fragments, and proIL-1β (34 kD) and mature (17 kD) fragments in the lysates and concentrated supernatants of J774A.1 cells primed for 4 h with 1 μg/ml LPS and stimulated with 3 mM ATP for 30 min with or without pretreatment with 500 μM MitoTEMPO. (d) Assessment of lactate dehydrogenase (LDH) activity in the supernatants of J774A.1 cells primed with 1 μg/ml LPS and stimulated with ATP for 30 min with or without pretreatment with 500 μM MitoTEMPO. Results are fold-change versus LPS primed cells and error bars represent standard error of two independent experiments. ** Indicates P<0.01 by one-way ANOVA with Tukey's post hoc comparison. (e and f) J774A.1 cells were left untreated, primed with LPS for 4 h, or primed with LPS and treated with 3 mM ATP as indicated. MitoSOX (red) was added to all samples 15 min after ATP addition and incubated for an additional 15 min before live-cell imaging. (e) Evaluation of the role of P2X7 and potassium efflux in mROS generation. 130 mM KCl and 25 μM A438079 were added 15–20 min before ATP addition. (f) Evaluation of the role of calcium influx in mROS generation. 100 μM BAPTA-AM was added 15–20 min before ATP addition. Nuclei are stained with Hoechst 33342 (blue). Scale bar is 50 μm. Results are representative of at least two independent experimentsCC BY · PubMed Central
Figure 7 Real-time, multiplexed visualization of ATP-induced potassium and calcium dynamics. J774A.1 cells were primed for 4 h with 1 μg/ml LPS, stained with KS6 and Fluo-4 DIRECT and both fluorophores were imaged simultaneously by confocal microscopy. (a) Representative fields for each condition showing Fluo-4 and KS6 signal responses. 16-color pseudocolor look-up tables were used for visualization. Blue indicates low signal intensity and red indicates high signal intensity. Scale bar represents 25 μM. (b) Mean and standard error for 30 representative cells in each condition. Where indicated cells were stimulated with 3 mM ATP. Inhibitors were added 15–20 min before imaging. Results are representative of at least two experiments
Figure 7 Real-time, multiplexed visualization of ATP-induced potassium and calcium dynamics. J774A.1 cells were primed for 4 h with 1 μg/ml LPS, stained with KS6 and Fluo-4 DIRECT and both fluorophores were imaged simultaneously by confocal microscopy. (a) Representative fields for each condition showing Fluo-4 and KS6 signal responses. 16-color pseudocolor look-up tables were used for visualization. Blue indicates low signal intensity and red indicates high signal intensity. Scale bar represents 25 μM. (b) Mean and standard error for 30 representative cells in each condition. Where indicated cells were stimulated with 3 mM ATP. Inhibitors were added 15–20 min before imaging. Results are representative of at least two experimentsCC BY · PubMed Central
Figure 8 Proposed mechanism for ion flux-dependent regulation of the NLRP3 inflammasome. Activation of the P2X7 receptor pathway with extracellular ATP results in the exchange of potassium and calcium across the plasma membrane, dominantly regulated by efflux of potassium from the cytosol to the extracellular space. Influx of calcium causes a mitochondrial calcium overload resulting in mitochondrial destabilization and mROS generation, which activates the NLRP3 inflammasome through an unknown mechanism, but possibly by involvement of TXNIP or Syk.6, 12, 16, 38, 39 P2X7 receptor activation also results in a mitochondrial potassium efflux that may be involved in mitochondrial destabilization and mROS generation. Inhibition of potassium efflux prevents calcium influx and downstream mROS generation. Likewise, chelation of intracellular calcium prevents mROS generation
Figure 8 Proposed mechanism for ion flux-dependent regulation of the NLRP3 inflammasome. Activation of the P2X7 receptor pathway with extracellular ATP results in the exchange of potassium and calcium across the plasma membrane, dominantly regulated by efflux of potassium from the cytosol to the extracellular space. Influx of calcium causes a mitochondrial calcium overload resulting in mitochondrial destabilization and mROS generation, which activates the NLRP3 inflammasome through an unknown mechanism, but possibly by involvement of TXNIP or Syk.6, 12, 16, 38, 39 P2X7 receptor activation also results in a mitochondrial potassium efflux that may be involved in mitochondrial destabilization and mROS generation. Inhibition of potassium efflux prevents calcium influx and downstream mROS generation. Likewise, chelation of intracellular calcium prevents mROS generationCC BY · PubMed Central

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  1. Murakami T, Ockinger J, Yu J, Byles V . Critical role for calcium mobilization in activation of the NLRP3 inflammasome. Proc Natl Acad Sci USA 2012; 109: 11282–11287. · DOI

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Authors
Yaron, J. R., Gangaraju, S., Rao, M. Y., Kong, X., Zhang, L., Su, F., Tian, Y., Glenn, H. L., & Meldrum, D. R.
Affiliations
Arizona State University (US)
Corresponding author
Meldrum, D R
ORCID
Venue
Cell Death & Disease
Publisher
Springer Science and Business Media LLC
Published
2015-10-29
Volume
6
Issue
10
Pages
e1954-e1954
ISSN
2041-4889 (ISSN-L)
DOI
10.1038/cddis.2015.277
PMC
PMC5399176
OpenAlex
W2212334792
Cited by
212
References
44
Citations by year
2026: 11 · 2025: 22 · 2024: 20 · 2023: 25 · 2022: 21 · 2021: 22 · 2020: 25 · 2019: 18 · 2018: 23 · 2017: 14 · 2016: 11
FWCI
6.94
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Death and Disease (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health
  • National Human Genome Research Institute (P50HG002360)
  • National Cancer Institute (U01CA164250)
  • Common Fund
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Inflammasome and immune disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Adenosine and Purinergic Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
Keywords
P2X7 receptor, NLRP3 inflammasome, potassium efflux, calcium influx, Fluo-4, mitochondrial reactive oxygen species, pyroptosis, macrophages, LPS priming, extracellular ATP, BAPTA-AM
MeSH terms
Adenosine Triphosphate, Animals, Calcium, Cell Line, Macrophages, Mitochondria, Models, Biological, Potassium, Reactive Oxygen Species, Calcium Signaling, Mice, Receptors, Purinergic P2X7, Inflammasomes

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