Research ArticleCell Biology

Chemical synapses without synaptic vesicles: Purinergic neurotransmission through a CALHM1 channel-mitochondrial signaling complex

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Science Signaling  08 May 2018:
Vol. 11, Issue 529, eaao1815
DOI: 10.1126/scisignal.aao1815
  • Fig. 1 Depolarization-evoked Ca2+ signals in type II taste cells.

    (A) Preparation for simultaneous monitoring of intracellular Ca2+ and ATP release. (B) Calcium responses (black dots; means ± SD; n = 7 cells; see table S1 for details) and voltage dependence of ATP release [red triangles; data from Romanov et al. (22)]. AU, arbitrary units. (C and D) Representative of recordings showing the effect of depolarization on (C) intracellular Ca2+ (n = 23 cells) and (D) voltage-related Ca2+ signals (n = 17 cells). Black arrows indicate Ca2+ responses, whereas the red line shows ATP sensor responses. (E) Effect of altering extracellular calcium on Ca2+ responses. Data are representative of five cells for each alteration. (F) Effect of decreasing extracellular pH on currents and Ca2+responses. Data are representative of three cells. (G) Effect of applying extracellular 100 μM Gd3+ (gray bar) on current and Ca2+ responses (n = 4 cells). P < 0.001, two-way ANOVA. Bottom trace shows control preparations. (H) Effect of applying negative pressure through the patch pipette on calcium events. Data are representative of three of five cells.

  • Fig. 2 CALHM1 lies at points of contact between taste cells and nerve fibers.

    (A to E) Immunostaining for CALHM1 (red), nerve fibers (P2X2; green), and type II taste cells (TrpM5-driven GFP; blue). Scale bars, 10 μm (A) and 5 μm [(B) to (E)]. Data are representative of multiple taste buds from each lingual papilla in more than five animals. Zeiss Airyscan images are shown in (D) and (E) (fig. S2); others are conventional laser scanning confocal microscope. Data are representative of four Airyscan images. (F) A line plot of a point of contact shown in (E). See also fig. S2. CALHM1 staining, red curve; type II cell cytoplasm, blue curve; nerve membranes, bimodal green line.

  • Fig. 3 CALHM1 lies adjacent to large mitochondria.

    (A to C) Colocalization of CALHM1 (red) and CytC (green) marking mitochondria. Type II taste cells (TrpM5-driven GFP) are rendered in blue. Scale bars, 2 μm [(A) and (B)] and 10 μm (C) with insets at 2× digital magnification and Unsharp Mask of 7.5 pixels at 85%. See fig. S1 for validation of the CALHM1 antibody. Data are representative of multiple taste buds from each lingual papilla in more than five animals. (D and E) Electron micrographs of a taste cell–neurite contact immunoreacted for CALHM1 with a peroxidase-based detection system. (E) An enlargement of the boxed area in (D). Scale bars, 500 nm (D) and 200 nm (E). The nerve fiber process is shaded yellow in (D) to facilitate visualization of the different structures in this unstained section. Data are representative of three taste cells in the CV papilla of one animal.

  • Fig. 4 Reconstructions of typical mitochondria from sbfSEM data.

    (A) Type II taste cell (green), a nerve fiber (yellow), and a type I cell (gray) showing the relationship of atypical mitochondria (red) and sensory nerve fibers. Data are representative of 29 type II taste cells in three CV taste buds from two mice. See movie S1. (B) Reconstruction of a type II taste cell (pale green; nucleus, dark green) showing typical (blue) and atypical (red) mitochondria in relation to the sensory nerve fiber (yellow). Scale bar, 10 μm. Data are representative of three reconstructed cells. See movie S2. (C) Enlarged bracketed region from (B), showing separation of atypical (red) from typical (blue) mitochondria. Scale bar, 1 μm. See movie S3. (D) Single image from an sbfSEM data set showing atypical mitochondria (red) and the afferent nerve terminal (yellow). Boxed area enlarged at right showing the tubular cristae within the atypical mitochondria. Scale bars, 1 μm. Data are representative of greater than 100 identified atypical mitochondria. (E and F) 3D reconstructions of the pair of typical (E) and atypical (F) mitochondria in (D). See movie S4.

  • Fig. 5 Cell membrane specialization at the CALHM1-mitochondrial complex.

    (A) Conventional transmission electron micrograph illustrating the relationship between the membrane of the atypical mitochondrion, the plasma membrane of the taste cells, and the nerve ending. Inset on the lower right is an enlargement of the boxed area in the main image. The inset has been enlarged (3×), and the edges has been enhanced by application of an Unsharp Mask filter (60% at five pixels). The arrow points to the synaptic cleft between the taste cell and nerve fiber. Scale bars, 100 nm for the main image and 33.3 nm for the inset. Data are representative of a minimum of five taste buds in CV papillae of more than five mice. (B) 3D reconstruction of a contact between type II taste cell and the nerve ending (yellow), including visualizations of conventional mitochondria in the nerve ending (magenta), and an atypical mitochondrion (red) and conventional mitochondria (cyan) inside the taste cell (green). Putative submitochondrial active zone is highlighted in blue. Detail to the right shows the method for approximating the volume of the different synaptic compartments.

  • Fig. 6 Blocking ATP production in mitochondria blocks release of ATP.

    (A) ATP biosensor cells were CHO cells transfected with mRNAs encoding P2X2 and P2X3 ionotropic purinergic receptors, which are responsive to ATP but not to ADP. (B) Depolarization-evoked ATP release from taste cells in the absence (blue trace) or presence (red trace) of oligomycin. The bar graph shows a quantitative comparison of the responses (5 min with oligomycin; n = 4 experiments with individual taste cells; means ± SEM; *P < 0.05). (C) ATP/ADP biosensor cells were COS-1 cells, which endogenously express P2Y receptors and are responsive to both ATP and ADP. (D) Depolarization-evoked release of ATP and ADP from taste cells in the absence (blue trace) or presence (red trace) of oligomycin. The bar graph shows a quantitative comparison of the responses (5 min with oligomycin; means ± SEM; n = 6 experiments with individual taste cells; *P < 0.05, t test). Scale bars, 10 μm [(A) and (C)]. Validation of biosensor sensitivity and functionality in the presence of oligomycin given in fig. S3.

  • Fig. 7 Schematic diagram of a mitochondrial/CALHM1 synapse between a type II taste cell and an afferent nerve terminal.

    The CALHM1 channels lie within the plasma membrane of the taste cell (light blue) between the atypical mitochondrion (pink) and the nerve terminal (green. (A) In the resting state, CALHM1 channels (blue) are closed and ATP levels in the presynaptic compartment will be close to levels in the intermembrane space of the mitochondrion due to diffusion of ATP (red circles) through the porin channels of the outer mitochondrial membrane. (B) Taste evoked action potentials in the taste cell open the CALHM1 channels, allowing ATP to flow into the synaptic cleft between the taste cell and the afferent nerve terminal. (C) The ATP in the intercellular space binds to and gates open the P2X receptors (green bars) in the neural membrane, permitting influx of sodium ions (Na+), which depolarizes the afferent terminal. (D) As the taste cell repolarizes, CALHM1 channels close and extracellular levels of ATP return to prestimulus levels.

  • Table 1 Measurements (in pixels) of mitochondrial features.

    Typical and atypical mitochondria were compared in three different general size classes: small, medium, and large.

    Intermembrane
    space
    Crista wallsCircumferenceEnclosed area
    (ECSA)
    Intermembrane
    space/total ECSA
    Crista walls/total
    ECSA
    Small
      Atypical2770.0 ± 224.5804.0 ± 53.5426.0 ± 24.76968.0 ± 467.20.40 ± 0.020.12 ± 0.01
      Typical859.0 ± 95.8727.0 ± 76.0509.5 ± 28.86781.0 ± 449.30.12 ± 0.010.11 ± 0.01
    Medium
      Atypical4368.0 ± 242.91460.0 ± 79.8673.8 ± 21.911,983.0 ± 521.60.37 ± 0.010.12 ± 0.01
      Typical1501.0 ± 121.31407.0 ± 101.1773.7 ± 39.812,371.0 ± 638.10.12 ± 0.010.11 ± 0.01
    Large
      Atypical13,461.0 ± 1014.73478.0 ± 246.91176.4 ± 62.837,254.0 ± 2726.60.37 ± 0.020.10 ± 0.00
      Typical2435.0 ± 264.62213.0 ± 201.01382.8 ± 53.421,849.0 ± 1118.70.11 ± 0.010.10 ± 0.01
    Overall meanAtypical0.38*** ± 0.005
    Typical0.12*** ± 0.003

    ***P < 0.0001, t test. All values ± SEM.

    Supplementary Materials

    • www.sciencesignaling.org/cgi/content/full/11/529/eaao1815/DC1

      Fig. S1. Validation of the CALHM1 antibody.

      Fig. S2. Localization of CALHM1 immunoreactivity in taste buds.

      Fig. S3. Calcium responses in sensor cells.

      Fig. S4. Effect of CBX on Mito-ID fluorescence in living taste cells.

      Table S1. Amplitudes of calcium responses in type II taste cells from Fig. 3B.

      Movie S1. 3D visualization of the relationship between a type II taste cell and the innervating nerve fiber shown in Fig. 1A.

      Movie S2. 3D visualization of mitochondria and taste cell shown in Fig. 1B.

      Movie S3. Relationship of atypical and typical mitochondria.

      Movie S4. Tubular cristae in atypical mitochondria.

    • Supplementary Materials for:

      Chemical synapses without synaptic vesicles: Purinergic neurotransmission through a CALHM1 channel-mitochondrial signaling complex

      Roman A. Romanov, Robert S. Lasher, Brigit High, Logan E. Savidge, Adam Lawson, Olga A. Rogachevskaja, Haitian Zhao, Vadim V. Rogachevsky, Marina F. Bystrova, Gleb D. Churbanov, Igor Adameyko, Tibor Harkany, Ruibiao Yang, Grahame J. Kidd, Philippe Marambaud, John C. Kinnamon, Stanislav S. Kolesnikov,* Thomas E. Finger*

      *Corresponding author. Email: tom.finger{at}ucdenver.edu (T.E.F.); staskolesnikov{at}yahoo.com (S.S.K.)

      This PDF file includes:

      • Fig. S1. Validation of the CALHM1 antibody.
      • Fig. S2. Localization of CALHM1 immunoreactivity in taste buds.
      • Fig. S3. Calcium responses in sensor cells.
      • Fig. S4. Effect of CBX on Mito-ID fluorescence in living taste cells.
      • Table S1. Amplitudes of calcium responses in type II taste cells from Fig. 3B.
      • Legends for movies S1 to S4

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Movie S1 (.mp4 format). 3D visualization of the relationship between a type II taste cell and the innervating nerve fiber shown in Fig. 1A.
      • Movie S2 (.mp4 format). 3D visualization of mitochondria and taste cell shown in Fig. 1B.
      • Movie S3 (.mp4 format). Relationship of atypical and typical mitochondria.
      • Movie S4 (.mp4 format). Tubular cristae in atypical mitochondria.

      © 2018 American Association for the Advancement of Science

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