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

Animals

Mice were utilized in all experiments involving live animals. All experimental protocols were in accordance with local regulatory bodies: the European Communities Council Directive (86/609/EEC) and approved by the regional ethical committee (Stockholms Norra Djurförsöksetiska Nämnd; N512/12) and by the Animal Care Committee of the Institute of Cell Biophysics, Pushchino; or by the local Animal Care and Use Committees at the The Feinstein Institute for Medical Research, North Shore LIJ Health System or at the Univ. Colorado School of Medicine. Particular effort was directed to minimize the number of animals and their suffering during the experiments. Tissue preparation, immunohistochemistry and imaging Wild-type and TrpM5-GFP (Tg(Trpm5-EGFP) #Sdmk ) transgenic mice (to reveal Type II taste cells) were perfused with a fixative composed of 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde in 0.1M phosphate buffer (PB, pH7.4) that was preceded by a short rinse with physiological saline (anesthesia: 5% isoflurane or Fatal-Plus Solution® [pentobarbital] Vortech Pharmaceuticals, Dearborn, MI,). After post-fixation in the same fixative 3 hr - overnight and cryoprotection in 10%–20% sucrose for 12 – 48h, tongues were cryo-sectioned at 12 – 20 μm thickness and dried onto positively-charged glass slides. In some specimens, antigen retrieval was carried out either using Dako solution (Dako S1699) or 10mM sodium citrate (pH 9 for 10 minutes at 85°C) prior to exposure to antisera to enhance antigen accessibility. Other sections were exposed to antibodies without antigen retrieval. Sections were then exposed for 16 – 72h at 4 °C to select combinations of primary antibodies diluted in PB or PBS to which 0.1 – 2% normal donkey serum and 0.3% Triton X-100 had been added. After extensive rinsing in PB, antibodies were revealed by fluorescent secondary antibodies 2h at 22 – 24 °C: donkey anti-rabbit and anti-mouse (carbocyanine (Cy)2, 3 or 5-tagged at 1:200; Jackson Immuno Research Laboratories; West Grove, PA); donkey anti-mouse A568 (Life Technologies; Carlsbad, CA); goat anti-rabbit A649 or donkey anti-chicken A488 (Jackson) at 1:400 dilution each). After 3 additional washes, sections were coverslipped with glycerol-based solution or with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL). Primary antibodies employed were: mouse monoclonal antibody 32C2 directed against the C-terminal domain of CALHM1 at 1:50 dilution ( 51 ); rabbit polyclonal antibody against cytochrome C (Santa Cruz, sc-7159; AB_2090474); rabbit anti-P2X3 at 1:1000 dilution (APR-016; Alomone Labs; Jerusalem, Israel; AB_2341047); chicken anti GFP at 1:1000 dilution (Aves Labs; Tigard, OR; AB_10000240). All staining reported herein was absent when the primary antibody was omitted. For ultrastructural immunohistochemistry, tissues were fixed as above, and then sectioned on a vibratome at 80μm and collected in phosphate buffer. Sections containing taste buds were incubated with 1% NaBH 4 for 10 minutes, rinsed and then transferred to 10 mM sodium citrate (pH 6) and 0.05% Tween 20 for 10 min at 85 °C. Afterwards, sections were placed for 15 mins. into an avidin-biotin blocking system involving exposure of the tissue first to Avidin D followed by incubation in unlabeled biotin (Vector SP-2001) containing 2% normal donkey serum plus AB media, then exposed to unlabeled goat anti mouse IgG Fab (1:25, Cat. 115-007-003, Jackson ImmunoResearch) for 1 hour to block non-specific binding of secondary antisera to endogenous IgG. The sections then were incubated with primary antibody monoclonal CALHM1 (1:20) for 4 nights. After washing, the sections were incubated overnight to biotinylated rat anti mouse IgG2a (1:1000, Cat. 04-6240, Life Technologies), then incubated with ABC (Vector Laboratories, Burlingame, CA) for two hours. The sections were treated for 10 min in 0.05 M Tris Buffer (pH 7.3) containing 0.05% DAB. The label was visualized by floating the sections for 2–4 min in the fresh DAB mixture with hydrogen peroxide (0.002%). Images were acquired on either an Olympus Fluoview Confocal microscope or on Zeiss LSM 700, LSM 710 or LSM 780 confocal laser-scanning microscopes with maximal signal separation or spectral scanning. Some images of Fig. 2 were processed with Zeiss Airyscan ™ . Composite figures were assembled in CorelDraw X5 or Photoshop CC 2014 (Adobe).

32C2 antibody specificity assessment by epitope blocking

Peptide array for epitope mapping was employed to determine the exact epitope sequence of the mouse IgG2a monoclonal antibody (32C2) against CALHM1. Ten-residue-long peptides, with an offset of 3 residues covering the entire cytosolic C-terminal end of human CALHM1, were dotted on nitrocellulose membrane. The membrane was then processed for Western Blot using 32C2, as previously described ( 51 ). The identified epitope peptide was used for antibody blocking. HT-22 cells were transiently transfected with empty vector or human CALHM1. Protein extracts were then processed by SDS-PAGE and transferred on nitrocellulose membranes. 32C2, pre-incubated 1h at room temperature with the indicated peptides (0.2 μg/mL) in 5% milk tween-TBS, was then used for WB. Serial Blockface Scanning EM (sbfSEM) and Transmission electron microscopy Methods for 3D-EM imaging and volumetric analysis are slightly modified from those described previously ( 52 , 53 ). Mice were anesthetized with Fatal-Plus Solution® and perfused with 0.1% NaNO 2 , 0.9% NaCl, and 200 units sodium heparin in 100 ml 0.1 M phosphate buffer pH7.3, at 35°C followed by 2.5% glutaraldehyde and 2% formaldehyde with 2mM CaCl 2 in 0.025 M cacodylate buffer pH7.3 at 35° C for 10 minutes. Tissues are removed and placed in the same fixative for 2–3 hours on ice, then cut into 200 μm thick vibratome sections. For conventional transmission EM, some thick sections prepared as for sbfSEM, were rinsed in buffer, then stained with 2% osmium tetrooxide in 0.05 M Sodium Cacodylate Buffer for 30 min. After rinsing, the sections were placed overnight in 1% uranyl acetate in double-distilled H 2 O and then stained en bloc in Walton’s lead at 60°C for 40 min prior to embedment in Luft’s Epon. Thin sections (90–120 nm) were cut with a diamond knife on a Reichert Ultracut E ultramicrotome, examined with FEI Tecnai G2 Biotwin Transmission Electron Microscope, photographed with Gatan Ultrascan 1000 digital camera. Vibratome sections (200 microns thickness) for sbfSEM were rinsed with 0.025 M cacodylate buffer pH7.3 containing 2mM CaCl 2 , then incubated for 1 hour at 0°C in a solution containing 3% K4[Fe(CN)6] in 0.025M cacodylate buffer pH7.3 with 2mM CaCl 2 combined with an equal volume of 4% aqueous OsO 4 . After the first heavy metal incubation, the sections are washed with H 2 O at room temperature 5×3 min. then placed in 1% thiocarbodydrazide solution for 20 min at room temperature. After washing, the sections are placed in 2% OsO 4 for 30 min at room temperature. Following this second exposure to osmium, the tissues are washed in H 2 O 5×3 min at room temperature, then placed in 1% (UO 2 (CH 3 COO) 2 ·2H2O) and left in a refrigerator overnight. The next day, en bloc Walton’s lead aspartate staining is performed for 30 min at 60°C in 0.066 g of Pb(NO 3 ) 2 in 10 ml of aspartic acid stock and pH adjusted to 5.5 with 1N KOH. Sections are dehydrated using an increasing series of ice-cold alcohol solutions before transfer into propylene oxide 5×3 min. and final embedment in Lufts Epon 3:7 at 60°C overnight. The tissues are then trimmed and mounted on an aluminium pin, coated with colloidal silver paste around the block edges, and then examined in a Zeiss Sigma VP system equipped with a Gatan 3View in-chamber ultramicrotome stage with low-kV backscattered electron detectors optimized for 3View systems. Samples are routinely imaged at 2.25kV, at 5–10nm/pixel resolution (30μm aperture, high current mode, high vacuum), with field sizes between 80–250 μm in x,y and approximately 500 slices with 80 nm thickness were generated. The resulting image stacks are aligned and montaged in Photoshop® (Adobe Systems) and ImageJ. Segmentation and reconstruction is accomplished using Reconstruct software.

Measurement of Mitochondrial Intermembrane Space

Atypical and typical mitochondria were selected from virtual sections derived from sbfSEM image stacks as viewed in Reconstruct. These mitochondrial types were distinguished based on several morphological features including overall size of the mitochondrion and exaggerated tubular shape of the cristae. Images were imported into Amira 5.6.0 (FEI Company, Hillsboro, Oregon) for volumetric analysis. A total of 20 atypical mitochondria and 18 typical mitochondria from a total of 3 different taste buds were analyzed. Typical mitochondria were selected to match approximately in terms of overall profile area of the atypical mitochondria measured. Either 3 or 4 profiles were analyzed per mitochondrion according to total number of profiles through the particular mitochondrion. Using the “MaterialStatistics” measurement function, we measured the total area of each mitochondrial profile as well as the area of the intermembrane space including the area within the tubular cristae. We then calculated the ratio of intermembrane space to total mitochondrial area for each mitochondrial profile. Within each class of mitochondrion, typical and atypical, no significant differences existed between samples or for different mitochondrial sizes. Accordingly data from the different sizes of mitochondria were pooled for a statistical comparison using an unpaired t-test on Graphpad ( www.graphpad.com/quickcalcs )..

Taste cell isolation

For isolation of taste buds or individual cells, 8–10 weeks old C57Bl6 mice were euthanized with CO 2 followed by cervical dislocation before tongues were removed. Taste cells were isolated from mouse (NMRI, 6–8-week old) circumvallate (CV) papilla. A tongue was injected between the epithelial and muscle layers with 0.7 mg/ml collagenase B, 1 mg/ml dispase II, 0.2 mg/ml elastase (all from Roche Diagnostics), and 0.5 mg/ml trypsin inhibitor (Sigma-Aldrich) dissolved in a solution (mM): 140 NaCl, 20 KCl, 0.3 MgCl 2 , 0.3 CaCl 2 , 10 HEPES-NaOH (pH 7.4). The tongue was incubated in an oxygenated Ca-free solution (in mM): 120 NaCl, 20 KCl, 1 MgCl 2 , 0.5 EGTA, 0.5 EDTA, 10 HEPES-NaOH (pH 7.4) for 20–30 min. The epithelium was then peeled off from the underlying muscle, pinned serosal side up in a dish covered with Sylgard resin, and incubated in the Ca-free solution for 10–30 min. The isolated epithelium was kept at room temperature in a solution (mM): 130 NaCl, 10 NaHCO 3 , 5 KCl, 1 MgCl 2 , 1 CaCl 2 , 10 HEPES-NaOH (pH 7.4), 5 glucose, 2 Na-pyruvate). Taste cells were removed from the CV papilla by gentle suction with a firepolished pipette with an opening of 70–90 μm and then expelled into an electrophysiological chamber. ATP and ATP/ADP biosensors and Calcium imaging Cells of the COS-1 line that endogenously express P2Y receptors coupled to Ca 2+ mobilization and CHO, were transfected with P2X2/P2X3 expression construct and used as cellular sensors for monitoring ambient nanomolar ATP and ADP concentrations. The bath solution for cellular physiology experiments contained (mM) 140 NaCl, 2.5 KCl, 1 MgSO 4 , 1.3 CaCl 2 , 1.2 NaH 2 PO 4 , 10 glucose, 5 pyruvate, 10 HEPES–NaOH, pH 7.4. For calcium imaging ATP-sensitive cells were preloaded with 4 mM Fluo-4AM+1.5 mg/ml Pluronic (both from Molecular Probes) for 30 min at 23–25°C. Cell fluorescence was excited with a computer controlled light emitting diode (Luxion) at 480 nm and recorded at 535 nm. Sequential fluorescence images were acquired every 0.5–2 seconds using a fluorescent Axioscope-2 microscope, an EMCCD Andor iXON camera (Andor Technology) and Workbench 6.0 software (INDEC Biosystems). Cells were stimulated by bath application of compounds. All chemicals were from Sigma-Aldrich. Experiments were carried out at 23–25°C.

Electrophysiology and calcium imaging

Taste cells were assayed with the patch-clamp technique using the perforated patch (with 400 mg/l amphotericin B in the recording pipette) or whole-cell configuration. Ion currents were recorded, filtered, and analyzed using an Axopatch 200B amplifier, a DigiData1322 interface, and the pClamp8 software (Axon Instruments). Intracellular solution contained (mM) 100 CsCl, 40 KCl, 1 MgATP, 1 EGTA, 10 HEPES–NaOH, pH 7.4. The bath solution contained (mM) 140 NaCl, 2.5 KCl, 1 MgSO 4 , 1.3 CaCl 2 , 1.2 NaH 2 PO 4 , 10 glucose, 5 pyruvate, 10 HEPES–NaOH, pH 7.4. For calcium imaging cells were loaded with 4 μM Fluo-4AM or FURA-2AM +1.5 mg/ml Pluronic (both from Molecular Probes) for 30 min at 23–25°C. For Fluo-4 loaded cells fluorescence was excited with a computer controlled light emitting diode (Luxion) at 480 nm and recorded at 535 nm. Sequential fluorescence images were acquired every 0.5–2 seconds using a fluorescent Axioscope-2 microscope, an EMCCD Andor iXON camera (Andor Technology) and Workbench 6.0 software (INDEC Biosystems). To measure Fura-2 signals, recordings were done using a VisiChrome monochromator and VisiView software (Visitron Systems) on an AxioExaminer.D1 microscope (Zeiss) equipped with a CoolSnap HQ 2 camera (Photometrics). Cells were stimulated by bath application of compounds. All chemicals were from Sigma-Aldrich. Experiments were carried out at 23–25°C.

Statistical analysis

Physiological data were analyzed using SigmaPlot (Systat Software Inc.). Data were expressed as means ± s.e.m. A p value of < 0.05 was considered statistically significant, and calculated by Student’s t -test or Mann-Whitney rank sum test.

Supplementary Material S1 Video Movie S1. 3D visualization of the relationship between a Type II taste cell and the innervating nerve fiber shown in Fig. 1A . S2 Video Movie S2. 3D visualization of mitochondria and taste cell shown in Fig. 1B . S3 Video Movie S3. Relationship of atypical and typical mitochondria S4 Video Movie S4. Tubular cristae in atypical mitochondria Supplementary Material Fig. S1. Validation of CALHM1 antibody. Fig. S2. Calcium responses of COS-1 sensor cells. Fig. S3. Effect of carbenoxolone on Mito-ID fluorescence in living taste cells.