Research ArticleCalcium signaling

L-type Ca2+ channel–mediated Ca2+ influx adjusts neuronal mitochondrial function to physiological and pathophysiological conditions

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Science Signaling  11 Feb 2020:
Vol. 13, Issue 618, eaaw6923
DOI: 10.1126/scisignal.aaw6923
  • Fig. 1 Effects of neuronal stimulation on the cytosolic ATP/ADP ratio and the cytosolic free Ca2+ concentration.

    The application of test solutions is indicated by color-coded horizontal bars. Oligomycin (Oligo) was coapplied during the experiment as indicated by the gray bar. Graphs show mean data points and SEMs. (A) Effect of changing the extracellular K+ concentration and of the subsequent application of oligomycin on the ATP/ADP ratio. n = 3 (4 mM K+), n = 5 (10 mM K+), n = 5 (20 mM K+), and n = 6 (10 mM K+ + BayK). (B) Effect of changing the extracellular K+ concentration and of the subsequent application of oligomycin on the ATP/ADP ratio in neurons pretreated with BAPTA-AM (n = 4 to 6). (C) Quantification and statistical evaluation of the change in ATP/ADP ratio (ΔATP/ADP) upon application of oligomycin in A and B (n = 4 to 8). ****P < 0.0001; *P = 0.01 to 0.05 [one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test]. (D) Effect of changing the extracellular K+ concentration and of the subsequent application of oligomycin on the ATP/ADP ratio in the presence of isradipine (Isra; n = 3 to 5). (E) Effect of application of glutamate (Glut) at the indicated concentrations and of subsequent oligomycin application on the ATP/ADP ratio. Isra was present in all experiments (n = 3 to 5). (F) Effect of 10 mM K+ stimulation with or without LTCC modulators on the cytosolic free Ca2+ concentration ([Ca2+]cyt). In the solution without a dihydropyridine, only the solvent DMSO was present. n = 9 (+DMSO, 10 mM K+ only); n = 6 (+BayK); n = 6 (+Isra). The asterisk indicates significant differences (P values between 0.01 and 0.05) between the data points outlined by square brackets, as revealed by t test. (G) Effect of 20 mM K+ stimulation with or without Isra (Isra) on the cytosolic free Ca2+ concentration ([Ca2+]cyt); n = 12 (+DMSO, 20 mM K+ only); n = 6 (+Isra). (H) Effect of glutamate application in the indicated concentrations in the presence of Isra on [Ca2+]cyt. n = 25 (30 μM glutamate) and n = 13 (100 μM glutamate). For (A) to (H), n represents the number of neurons investigated in at least three separate culture dishes.

  • Fig. 2 Effects of neuronal stimulation on Ψmt.

    (A) Effect of the application of the indicated test solutions (indicated by color-coded horizontal bars) on Ѱm. Oligomycin and FCCP were applied during the experiment as indicated by the gray bars (n = 5 to 10). (B) ΔΨmt in response to oligomycin in (A) were quantified and are shown with statistical evaluation in the scatterplot (n = 7 to 11). ****P < 0.0001; ***P = 0.0001 to 0.001; **P = 0.001 to 0.01; *P = 0.01 to 0.05 (one-way ANOVA with Bonferroni’s multiple comparisons test). (C) Effect of 10 mM K+ + BayK in the absence (green trace) (n = 4) or presence of preapplied oligomycin (gray trace) on Ψmt (n = 5). (D) Effect of the application of 1 μM bicuculline (Bic) without or with BayK or Isra and subsequent coapplication of oligomycin on Ψmt (n = 3 to 7). (E) Effect of the application of 10 μM bicuculline without or with BayK or Isra and subsequent coapplication of oligomycin on Ψmt (n = 3 to 7). The effect of low Mg2+ solution and subsequent coapplication of oligomycin is also shown in the purple trace. (F) Effect of 1 μM bicuculline + BayK in the absence (green trace) or presence of preapplied oligomycin (gray trace) (n = 4 for each trace). For (A) and (B), n represents the number of neurons investigated in three separate culture dishes. For (C) to (F), n represents the number of neurons investigated in two separate culture dishes.

  • Fig. 3 Effects of neuronal stimulation on mitochondrial free Ca2+ concentration, neuronal death, and mitochondrial morphology.

    (A) Mitochondrial Ca2+ signals induced by 10 mM K+ (black trace, n = 6) and 10 mM K+ + BayK (green trace, n = 6). (B) Mitochondrial Ca2+ signals induced by 20 mM K+ (blue trace, n = 3) and 20 mM K+ + Isra (orange trace, n = 6). (C) Mitochondrial Ca2+ signals induced by 10, 30, and 100 μM glutamate (in the presence of Isra; n = 3 to 4). In (A) to (C), n represents the number of neurons investigated in separate culture dishes. (D) Mitochondrial Ca2+ transients evoked by repeated application of 10 mM K+ + BayK in the same neuron (representative of three independent experiments performed in separate culture dishes). (E) Elicitation of a persistent mitochondrial Ca2+ elevation during the course of repeated application of glutamate (representative of five independent experiments performed in separate culture dishes). (F) Effect of RMO-inducing solutions on neuronal death determined in a PI assay (n = 5 separate culture dishes). ****P < 0.0001 (one-way ANOVA with Bonferroni’s multiple comparisons test). (G) Morphological changes in mitochondria fluorescently labeled with mito-DsRed upon application of 100 μM glutamate (representative of five independent experiments performed in separate culture dishes). (H) Morphological changes of mitochondria fluorescently labeled with mito-DsRed upon application of 10 mM K+ + BayK (representative of three independent experiments performed in separate culture dishes). Scale bars (G and H), 5 μm.

  • Fig. 4 Analysis of the involved intracellular signaling cascades.

    (A) Effect of 10 mM K+ and of subsequent coapplication of oligomycin on the ATP/ADP ratio in neurons pretreated with thapsigargin. The dotted gray line retraces the corresponding data from Fig. 1A in all graphs; the dashed line highlights the trend toward a relative increase during the later phase of the response (n = 3). (B) Effect of 10 mM K+ + BayK and of subsequent coapplication of oligomycin on the ATP/ADP ratio in neurons pretreated with thapsigargin (n = 3). (C) Scatterplot showing the oligomycin responses during stimulation with 10 mM K+ and 10 mM K+ + BayK in the presence (n = 3 in both cases) or absence (n = 8 and 7, respectively) of thapsigargin. **P = 0.001 to 0.01 (one-way ANOVA with Bonferroni’s multiple comparisons test). (D) Effect of 10 mM K+ and of subsequent coapplication of oligomycin on the ATP/ADP ratio in neurons pretreated with L-NAME (n = 4). (E) Effect of 10 mM K+ + BayK and of subsequent coapplication of oligomycin on the ATP/ADP ratio in neurons pretreated with L-NAME (n = 4). (F) Scatterplot showing the changes in the ATP/ADP ratio in response to oligomycin without (−) and with (+) L-NAME (as indicated) during stimulation with 10 mM K+ (−, n = 8; +, n = 4) and 10 mM K+ + BayK (−, n = 7; +, n = 4). ****P < 0.0001 (one-way ANOVA with Bonferroni’s multiple comparisons test). For (A) to (F), n represents the number of neurons investigated in separate culture dishes.

  • Fig. 5 Stimulation-induced changes in cytosolic and mitochondrial NO production.

    (A to D) Effect on the fluorescence of the geNOp probe targeted to the cytosol (Cyto) or the mitochondrial (Mito) matrix elicited by stimulation with 10 mM K+ (n = 3) (A), 10 mM K+ + BayK (n = 4) (B), 10 mM K+ + Isra (n = 6) (C), and 100 μM glutamate (n = 3) (D). For (A) to (D), n represents the number of neurons investigated in separate culture dishes.

  • Fig. 6 Analysis of mitochondrial Ca2+ elevations.

    (A) Changes in mitochondrial free Ca2+ and of free Ca2+ in the endoplasmic reticulum (ER) upon stimulation with 10 mM K+ (application indicated by the black horizontal bar) (n = 6). (B) Changes in mitochondrial free Ca2+ upon stimulation with 10 mM K+ in neurons pretreated with thapsigargin (n = 3). The dotted gray line retraces the data in (A) from neurons not treated with thapsigargin. (C) Changes in mitochondrial free Ca2+ and of free Ca2+ in the ER upon stimulation with 10 mM K+ + BayK (application indicated by the black horizontal bar) (n = 6). (D) Changes in mitochondrial free Ca2+ upon stimulation with 10 mM K+ + BayK in neurons pretreated with thapsigargin (n = 3). The dotted gray line retraces the data in (C) from neurons not treated with thapsigargin. (E) Effect of stimulation with 10 mM K+ + BayK on the concentration of mitochondrial free Ca2+ in the continuous presence of L-NAME (green trace, n = 3). (F) Effect of stimulation with 10 mM K+ + BayK on the concentration of mitochondrial free Ca2+ in the continuous presence of cyclosporine A (green trace, n = 4). The dotted gray lines in (E) and (F) retrace data from untreated neurons in (C) (red trace). In (A) to (F), n represents the number of neurons investigated in separate culture dishes.

  • Fig. 7 LTCC-dependent regulation of the mitochondrial ATP synthase in response to various neuronal discharge activities.

    (A) Glycolysis. (B) LTCC-mediated Ca2+ influx. (C) Ca2+-induced Ca2+ release (CICR). (D) Mitochondria-associated membranes (MAMs). (E) Mitochondrial carrier proteins (MCPs). (F) Pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (IDH), and α-ketoglutarate dehydrogenase (α-KGD). (G) Electron transport chain (ETC). (H) ATP synthase operating in forward mode. (I) Mitochondrial Ca2+ rise. (J) Mitochondria-associated nitric oxide synthase (NOS). (K) Nitric oxide (NO). (L) ATP synthase operating in reverse mode. (M) H+-driven Na+ extrusion and mitochondrial Na+/Ca2+ exchanger. (N) Mitochondrial permeability transition pore (mPTP). ATP, adenosine triphosphate; ims, intermembrane space; m, mitochondrial matrix; NAD+ and NADH, oxidized and reduced form of nicotinamide adenine dinucleotide.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/618/eaaw6923/DC1

    Fig. S1. Fluorescence images of cultured hippocampal neurons to demonstrate the subcellular localization of the fluorescent indicators.

    Fig. S2. Electrophysiological recordings of neuronal activity induced by K+ and glutamate.

    Fig. S3. Full recovery of the ATP/ADP ratio after drops caused by RMO-inducing stimulations.

    Fig. S4. Stimulation protocols used are not accompanied by considerable pH changes.

    Fig. S5. Exploring the specificity of Isra using the structurally unrelated LTCC inhibitor diltiazem.

    Fig. S6. Electrophysiological recordings of bicuculline- and low Mg2+-induced neuronal firing.

    Movie S1. Example of reversible mitochondrial fragmentation.

  • The PDF file includes:

    • Fig. S1. Fluorescence images of cultured hippocampal neurons to demonstrate the subcellular localization of the fluorescent indicators.
    • Fig. S2. Electrophysiological recordings of neuronal activity induced by K+ and glutamate.
    • Fig. S3. Full recovery of the ATP/ADP ratio after drops caused by RMO-inducing stimulations.
    • Fig. S4. Stimulation protocols used are not accompanied by considerable pH changes.
    • Fig. S5. Exploring the specificity of isradipine using the structurally unrelated LTCC inhibitor diltiazem.
    • Fig. S6. Electrophysiological recordings of bicuculline- and low Mg2+-induced neuronal firing.
    • Legend for movie S1

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Example of reversible mitochondrial fragmentation.

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