K gluconate

Patch clamp electrodes were pulled from borosilicate glass capillaries (1B120F-4, World Precision Instruments, Inc., Sarasota, FL, USA) with a horizontal puller (Model P-87, Sutter Instrument Co.). The electrode resistances were 5–8 MΩ when filled with the solution. The internal solution contained 115 mM K gluconate (Sigma), 4.42 mM KCl (Fisher), 10 mM Na₂ phosphocreatine (Sigma), 10 mM HEPES (Sigma), 0.5 mM EGTA (Sigma), 4 mM Mg-ATP (Sigma), 0.3 mM Na-GTP (Sigma) and 0.1 or 0.2% biocytin (Invitrogen), with pH 7.30 (adjusted with KOH, Sigma) and osmolality 300 mmol/kg (adjusted with sucrose, Sigma) (Roberts et al., 2014 (link)). Patch clamp recordings were obtained using the blind in vivo method as described before (Margrie et al., 2002 (link); Franken et al., 2015 (link)). Membrane potential recordings were obtained in current clamp using a patch clamp amplifier (BVC-700A; Dagan, Minneapolis, MN, USA). The analog signal was low-pass filtered (cut-off frequency 5 kHz), digitized at 50–100 kHz and saved using scripts in MATLAB (The Mathworks) or IgorPro (WaveMetrics). Series resistance was 51.7 ± 10.8 MΩ (mean ± SEM; N = 8; excluding one outlier with a series resistance >100 MΩ). Initial resting membrane potential was –54.6 ± 1.95 mV (mean ± SEM; N = 10).

A cover slip with cultured cells was transferred to a recording chamber (Warner Instruments) and placed under a microscope (Olympus). Artificial CSF, containing 124 mM NaCl, 3 mM KCl, 1.3 mM MgSO₄, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 2.4 mM CaCl₂-2H₂O, and 10 mM glucose (Sigma-Aldrich) was continuously perfused over the cells and aerated with O₂ 95%/CO₂ 5% mixed gas at RT. A glass capillary pipette was directed towards the cell surface. Negative pressure was provided to elicit a gigaseal for whole-cell recording. The internal pipette solution contained 115 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 10 mM EGTA, 5 mM Mg-ATP, and 0.5 mM Na²⁺-GTP (Sigma-Aldrich), with pH 7.3 and 280–285 mOsm. Sodium current was recorded in voltage clamp mode, and electrical stimulation was provided in the range −60 mV to +20 mV. Thereafter, current clamp mode was utilized to evaluate action potential generation. To verify that inward currents and spikes were mediated by voltage gated sodium channels, the bath was treated with tetrodotoxin (TTX; 0.5 μM, Sigma-Aldrich) prior to recording. Data acquisition was performed using Digitizer 1440A (Molecular Devices) and Clampex 10.3 (Molecular Devices). Analysis of data was conducted by using Clampfit 10.3 (Molecular Devices).

AlexaFluor-594 hydrazide, AlexaFluor-594 dextran (10,000 MW), Fluo-4 and Fluo-4FF pentapotassium salts were purchased from Invitrogen. CsCl and NaHCO₃ were obtained from ACP Chemicals, tetrodotoxin (TTX) from Alomone Labs, N-ethyllidocaine bromide (QX-314, Br-) and bicuculline from Tocris Bioscience, SP20 antibody from Santa Cruz Biotechnology (Cat# sc-65512, Lot# K1407, RRID:AB_1129364), paraformaldehyde from Electron Microscopy Sciences. We ordered NaCl, KCl, glucose, NaH₂PO₄, Na-pyruvate, myo-inositol, l-ascorbic acid, MgCl₂, CaCl₂, K-gluconate, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), HEPES, phosphocreatine, Na-ATP, Na-GTP, phosphocreatine di(tris) salt, tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP) and Strychnine from Sigma-Aldrich.

Cells were passaged one day prior to plating at a density of 2×10⁵ cells/mL in 5 or 10mL of regular growth media with an added 80mM K-gluconate (Sigma, P1847) or CaCl₂ (Sigma, C-3306) (unless otherwise noted). After indicated incubation times and depending on the experiment, RNA was either bulk harvested from treated cells or was harvested from live cells that were first sorted and collected by flow cytometry based on FSC-A and SCC-A measurements (indicated in figure legends). For pulse/chase in Fig 5E, 5F and 5G, cells were treated as indicated for 24 hours. 3×10⁵ live cells were then sorted and returned to culture followed by removal of aliquots at indicated time points for harvesting of RNA (0h = 24 hour pulse, 0 hour chase). Experiments for all figures were performed in triplicate (at minimum) unless otherwise noted in figure legends.

The cover slip with cultured cells was transferred to the recording chamber (Warner Instruments, Hamden, CT, USA) and placed on the microscope (Olympus, Tokyo, Japan) while continuously perfused with cerebrospinal fluid containing: 124 mM NaCl, 3 mM KCl, 1.3 mM MgSO₄, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 2.4 mM CaCl₂-2H₂O, and 10 mM glucose (all from Sigma-Aldrich). The solution was continuously aerated by O₂ 95 %/CO₂ 5 % mixed gas at RT.
A glass capillary pipette approached the cell surface. After that, negative pressure was provided to let the pipette go giga seal and whole-cell mode. Internal pipette solution contained 115 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 10 mM EGTA, 5 mM Mg-ATP, and 0.5 mM Na²⁺-GTP (all from Sigma-Aldrich), with pH 7.3 and 280–285 mOsm. Holding potential was −60mV. Na⁺ current was recorded in voltage clamp mode, and electrical stimulation was given with a range from −60 mV to +50 mV (+10 mV per each step). After that, cells went to current clamp mode for testing action potential generation. Cells received 15 steps of current (initial level = 0 pA, Δ3–10 pA per step) according to their membrane capacity. To verify that currents and spikes were mediated by Na⁺ channels, tetrodotoxin (TTX) (0.5 μM) (Sigma-Aldrich) was added to the bath for 5–10 min, and currents and spikes were retested.