Glass coverslip

We performed experiments on rat cultured hippocampal neurons, with approval for all animal procedures from the Yale University Animal Care and Use Committee. The methods to culture neurons are essentially the same as previously described (Brewer et al., 1993 (link)) and subsequently modified in the Boron laboratory (Chen et al., 2008 (link)). Briefly, a pregnant rat was deeply anesthetized using halothane, prior to cervical dislocation. Rat embryos (18 days) were quickly removed from the uterus and decapitated. Approximately ten brains were collected and placed in filtered HEPES-buffered solution (HBS) containing (in mM): NaCl, 143.7; KCl, 3; and HEPES, 10. Hippocampi were extracted using fine forceps and a scalpel and then exposed to 0.03% (w/v) trypsin (Sigma-Aldrich, St Louis, MO) dissolved in HBS for 15 min at 37°C. Using flamed Pasteur pipettes with reduced tip diameter, we triturated the tissues to disperse cells. Neurons were plated in neurobasal medium supplemented with B-27 (GIBCO-Invitrogen, Carlsbad, CA), 10% fetal calf serum (FCS), plus penicillin-streptomycin on poly-L-lysine (MP Biomedical, Irvine, CA) coated glass coverslips (Warner Instruments, Hamden CT, USA) or photoetched grid coverslips (Bellco Biotechnology, Vineland, NJ). After 3–4 h the medium was changed to a similar one without FCS. Neurons cultures were kept in a 5% CO₂-air incubator at 37°C for at least seven and up to 61 days (average 23 days).

Plasmids. Empty pCMV6-entry plasmid and pCMV6-entry plasmid encoding Mus musculus Slc4a8 Myc-DDK tagged (GenBank Accession no. NM_021530) and Slc4a10 Myc-DDK tagged (transcript variant 5, GenBank Accession no. NM_001242381) were obtained from Origene Technologies Inc. (Rockville, MD 20850, USA).
Cell culture and transfections. CHO-K1 cells (Sigma-Aldrich, Saint Louis, MO 63103, USA) were grown as previously described [11 (link)]. Cells were electroporated (Nucleofector II, Amaxa, Gaithersburg, MD 20877, USA) with the plasmids mentioned above (4 µg DNA per reaction) using the Nucleofector Kit V (Lonza, Morristown, NJ 07960, USA) following the manufacturer’s instructions and then seeded onto 5 mm diameter glass coverslips (Warner Instruments, Holliston, MA 01746, USA). The expression of Slc4a8 and Slc4a10 in CHO-K1 cells resulted in comparable yields: 55.7 ± 4.7% for Slc4a8 and 53.4 ± 4.9% for Slc4a10.
For the immunofluorescence experiments (see methods below), CHO-K1 cells were transfected with 2.5 μg of Slc4a8, Slc4a10 or empty plasmids using the Xfect (Takara, San Jose, CA 95131, USA) transfection reagent according to the manufacturer’s instructions.
Immunofluorescence experiments. CHO-K1 cells were trypsinized after transfections and seeded onto 25 mm diameter glass coverslips (Superior Marienfeld, 97922 Lauda-Königshofen, Germany). At 24 h after transfection, the cells were incubated for 60 min on ice in complete culture medium supplemented with wheat germ agglutinin (WGA) conjugated to Alexa Fluor 633 fluorophore (1 μg/mL, Thermo Fisher Scientific, Waltham, MA 02451, USA), followed by cell fixation with 4% paraformaldehyde (PFA) for 20 min and methanol for 45 min at −20 °C. The coverslips were air dried and then stored overnight at −80 °C. The next day, cells were blocked with 3% bovine serum albumin (BSA) diluted in phosphate-buffered saline (PBS) containing (in mM): 137 NaCl, 2.7 KCl, 8 Na₂HPO₄, 2 KH₂PO₄, at pH 7.4. After blocking, the coverslips were incubated for 1 h at room temperature (RT) with a mouse monoclonal anti-DYKDDDDK antibody (9A3, 1:500 dilution, Cell Signaling Technologies, Danvers, MA 01923, USA) followed by three 5 min washings with PBS. Then, the coverslips were incubated for 1 h at RT with goat anti-mouse IgG (H + L) conjugated to Alexa Fluor 488 (1:1000 Jackson Immunoresearch, West Grove, PA 19390, USA) followed by three washings with PBS. Finally, nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI 1:50,000 dilution, Thermo Fisher Scientific) and mounted using fluorescence mounting medium (Dako, Santa Clara, CA 95051, USA).
Images were acquired using a DMi8 Leica spinning disk confocal microscope equipped with a HC PL APO 63x×oil immersion objective (N.A.1.40) and an EMCCD digital camera (iXon Life 888, Andor Technology, Belfast, United Kingdom). Images were captured using SlideBook 6 software (Intelligent Imaging Innovations, Denver, CO 80216, USA) and processed with ImageJ software (version 1.54f) [23 (link)].
Intracellular pH, [Cl⁻] and [K⁺] measurements. The solutions used in this study are shown in Table 1 and Table 2. Functional experiments were conducted 18–20 h after electroporation. The fluorescent ion-sensitive dyes BCECF-AM (2′,7′-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester) [24 (link)], PBFI-AM (1,3-benzenedicarboxylic acid, 4,4′-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester) [16 (link)] and SPQ (6-methoxy-N-[3-sulfopropyl] quinolinium) [25 (link)] were purchased from Thermo Fisher Scientific and used for measuring intracellular pH, [K⁺] and [Cl⁻], respectively.
For intracellular pH measurements, CHO-K1 cells were loaded with 2 μM BCECF-AM for 15 min at 37 °C (95% O₂/5% CO₂). For intracellular K⁺ measurements, CHO-K1 cells were loaded with 10 μM PBFI-AM for 80 min at room temperature (95% O₂/5% CO₂). For intracellular [Cl⁻] measurements, CHO-K1 cells were exposed to a hypotonic loading solution containing 10 mM SPQ as previously described [26 (link)]. Solutions containing HCO₃⁻ were gassed with 95% O₂/5% CO₂. For experiments performed under HCO₃⁻-free conditions, solutions were gassed continuously with 100% O₂ and supplemented with 20 μM ethoxyzolamide (EZA), a carbonic anhydrase inhibitor.
Imaging experiments were performed using an inverted microscope (Olympus IX71, Olympus America Inc., Center Valley, PA 18034, USA) equipped with a Polychrome IV Imaging System coupled to a high-speed digital camera (Till Photonics, Victor, NY 14564, USA). Images were acquired by alternate excitation at 490 and 440 nm (BCECF), 340 and 380 nm (PBFI) or excitation at 340 nm (SPQ). Emissions were captured at 530 nm (BCECF) or 510 nm (PBFI and SPQ) using Imaging WorkBench 6.0 software (INDEC BioSystems, Los Altos, CA 94022, USA) or Micromanager 1.4 software [27 (link)]. The temperature of the solutions (listed in Table 1 and Table 2) was kept at 37 °C during the experiments using a CL-100 bipolar temperature controller (Warner Instruments).
Slc4a8 and Slc4a10 dependence on Na⁺ and K⁺ concentrations. The dependence of Slc4a8 and Slc4a10 for Na⁺ and K⁺ ions was obtained by fitting the normalized Slc4a8 and Slc4a10 activities (flow rates correspond to the slopes obtained by linear regression analysis of the linear response recorded when transitioning from an NMDG-containing external solution to either a Na⁺ or K⁺-containing external solution) measured at different [Na⁺]_o and [K⁺]_o to the following Hill function using Origin 8.0 software (OriginLab): $${\mathit{R}}_{\mathit{C}\mathit{a}\mathit{t}\mathit{i}\mathit{o}\mathit{n}}={\mathit{R}}_{\mathit{M}\mathit{i}\mathit{n}}+\frac{\left({\mathit{R}}_{\mathit{M}\mathit{a}\mathit{x}}-{\mathit{R}}_{\mathit{M}\mathit{i}\mathit{n}}\right)\times {\left[\mathit{C}\mathit{a}\mathit{t}\mathit{i}\mathit{o}\mathit{n}\right]}^{\mathit{n}\mathit{H}}}{{{\mathit{E}\mathit{C}}_{50}}^{\mathit{n}\mathit{H}}{+\left[\mathit{C}\mathit{a}\mathit{t}\mathit{i}\mathit{o}\mathit{n}\right]}^{\mathit{n}\mathit{H}}}$$
where R_Cation is the alkalinization rate in response to a reduction in external [Cl⁻] at different [Cation], and R_Min and R_Max correspond to the minimal and maximal alkalinization rates. EC₅₀ is the concentration required to achieve 50% of the maximal effect and nH is the Hill number.
Molecular dynamics simulations. The cryo-EM structure of the rat Slc4a8 (PDB: 7rtm) with K⁺ in place of Na⁺ at the ion coordination pocket was used as input for molecular dynamics (MD) simulations. A total of three MD simulations were performed for 100 ns each. They were executed using Desmond v2019-1 [28 ] and OPLS2005 force field [29 (link),30 (link),31 (link)]. The Slc4a8 structure with the K⁺-CO₃²⁻ pair at the S1 site was prepared using Protein Preparation Wizard from Maestro suite [32 (link)]. The system was embedded into a pre-equilibrated POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayer membrane model and solvated using the single point charge (SPC) water model. To neutralize the system, Na⁺/Cl⁻ ions were included; NaCl was added at a concentration of 0.15 M. The system was equilibrated for 20 ns in the NPT ensemble. Positional restraints of 1.0 kcal×mol⁻¹×Å⁻² were applied to the protein and to the ions placed in coordination sites (K⁺ and CO₃²⁻). Temperature and pressure were kept constant at 300 K and 1.01325 bar, respectively, by coupling to a Nose–Hoover Chain thermostat [33 (link)] and Martyna–Tobias–Klein barostat [34 (link)]. The integration step was set to 2 fs. For MD productive runs, the positional restraints were removed. Each run was performed during 100 ns using an NPγT (semi-isotropic ensemble) with a constant surface tension of 0.0 bar Å. For each trajectory, the root-mean-square deviation (RMSD) values of the backbone atoms were computed using VMD v1.9.4a38 [35 (link)]. The distances between ions and residues were computed using TCL scripting in VMD v1.9.4a38 [35 (link)].
Sequence alignment. Multiple sequence alignment was performed using Clustal Omega [36 (link)]. Sequences were retrieved from the UniProtKB database and correspond to mouse Slc4a8 (code: Q8JZR6), rat Slc4a8 (code: F1LUB7) and mouse Slc4a10 (code: Q5DTL9).
Statistical analysis. Results are presented as the mean ± standard error of mean (SEM), where n corresponds to the number of experiments per condition. Data were obtained from at least three independent electroporations (or transfections for immunolocalization studies) per condition. Statistical significance was determined using Student’s t test and one-way ANOVA analysis followed by Bonferroni’s post hoc test. p values of less than 0.05 were considered statistically significant. Origin 8.0 Software was used for statistical calculations (OriginLab, Northampton, MA, USA).