Mgcl2

The Hexokinase assay was performed as previously described^{87 (link)}. Briefly, 20 µg of fresh tissue lysate was added to 1 ml of reaction buffer for hexokinase (50 mM Tris HCl (Sigma-Aldrich), pH 7.5, 10 mM MgCl₂ (Sigma-Aldrich), 0.6 mM ATP (Sigma-Aldrich), 100 mM glucose (Sigma-Aldrich), 0.2 mM NADP+ (Sigma-Aldrich), and 0.1 units of glucose-6-phosphate dehydrogenase (Sigma-Aldrich)). Ten units of glyceraldehyde-3-phosphate dehydrogenase (Sigma-Aldrich) per ml was used for analyzing the Hexokinase activity. The Pyruvate kinase assay was performed as previously described^{87 (link)}. Briefly, 20 µg of fresh tissue lysate was added to 1 ml of reaction buffer for pyruvate kinase (50 mM Tris HCl (Sigma-Aldrich), pH 7.5, 5 mM MgCl₂ (Sigma-Aldrich), 5 mM ATP (Sigma-Aldrich), 0.2 mM NADH (Sigma-Aldrich) 100 mM KCl (Sigma-Aldrich), 5 mM Na₂HPO₄ (Sigma-Aldrich), 5 mM MgCl₂ (Sigma-Aldrich), 0.01 mM AMP (Sigma-Aldrich)) 5 mM fructose-6-phosphate (Sigma-Aldrich), 5 units of triosephosphate isomerase (Sigma-Aldrich) per ml, 1 unit of aldolase (Sigma-Aldrich) per ml was added to check the pyruvate kinase activity. A negative and positive control has been included without tissue lysate and with 0.05 units of hexokinase and pyruvate kinase in both the assays. Enzyme activities were measured and represented as the change in absorbance/min, calculated using a linear portion of the obtained curve.

The basal medium used for osteogenic induction was high-glucose Dulbecco's modified Eagle's medium (HG-DMEM) for mMSCs and Iscove's modified Dulbecco's medium (IMDM) for hMSCs supplemented with the induction factors: 0.1 μM dexamethasone (Sigma-Aldrich), 10 mM β-glycerol phosphate (βGP; Sigma-Aldrich), and 0.2 mM ascorbic acid (ASA; Sigma-Aldrich) [31 (link)]. The cells were seeded at 4000 cells/cm² and were maintained in culture medium (low-glucose DMEM supplemented with 10% FBS) for 24 hours before the induction. The induction medium was changed twice a week.
DMEM and IMDM contain 0.8 mM magnesium ion, and therefore 0.8 mM magnesium concentration is defined as normal magnesium concentration. The serum magnesium level in patients with renal disease is around 1 mM [32 (link)] and a serum magnesium level around 5 mM is associated with profound muscle weakness; on the other hand, physiological magnesium concentrations in soft tissue and bone are 8.5 and 43.2 mmol/kg wet weight respectively [15 (link)]. Therefore, for the induction medium with high concentration of magnesium, DMEM and IMDM were supplemented with 5 mM MgCl₂ (Sigma-Aldrich) and we defined 5.8 mM MgCl₂ as the experimental group of high magnesium concentration. A 1 M MgCl₂ stock solution was prepared in double-distilled water (ddH₂O) to make the final concentration of magnesium 5.8 mM.

Mice were briefly anesthetized with 3% sevoflurane before decapitation. Acute sagittal hippocampal slices (300 μm) were obtained using a VT1200S vibratome (Leica, Switzerland) in ice-cold dissection buffer (212 mM sucrose [Sigma-Aldrich, Cat# S5016], 25 mM NaHCO₃ [Sigma-Aldrich, Cat# S6297], 5 mM KCl [Sigma-Aldrich, Cat# P3911], 1.25 mM NaH₂PO₄ [Sigma-Aldrich, Cat# S0751], 10 mM D-glucose [Sigma-Aldrich, Cat# G7578], 2 mM sodium pyruvate [Sigma-Aldrich, Cat# P2256], 1.2 mM sodium ascorbate [Sigma-Aldrich, Cat# A4034], 3.5 mM MgCl₂ [Sigma-Aldrich, Cat# M0250], 0.5 mM CaCl₂ [Sigma-Aldrich, Cat# C3881], continuously aerated with 95% O2/5% CO2). The slices were transferred to a chamber filled with warmed (32 °C) artificial cerebrospinal fluid (aCSF: 125 mM NaCl [Sigma-Aldrich, Cat# S7653], 25 mM NaHCO₃, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 10 mM D-glucose, 1.3 mM MgCl₂, 2.5 mM CaCl₂, aerated with 95% O2/5% CO₂) for recovery (30 minutes), and then placed in room temperature prior to the experiments.

Purified SUR2B proteins were further supplemented with 3 mM fluorinated Fos-Choline-8 (FFC), 8 mM MgCl₂, 8 mM ATP (Sigma) and 200 μM P1075 (Tocris Bioscience) for Mg-nucleotides + P1075 state; 8 mM MgCl₂ and 8 mM ADP (Sigma) for Mg-nucleotides state; 8 mM MgCl₂, 8 mM ADP and 200 μM Lev (MedChemExpress) for Mg-nucleotides + Lev state. Purified SUR2A proteins were supplemented with 3 mM FFC, 8 mM MgCl₂, 8 mM ADP and 200 μM P1075 for Mg-nucleotides + P1075 state. Cryo-EM sample was loaded on to glow-discharged Quantifoil 0.6/1 gold grids and frozen as described previously^{19 (link)}.

Staining was performed on cryosections. Tissues were washed with 2 mM MgCl₂ in PBS twice for 5 min each at room temperature and incubated in X-gal (4%) mixed with X-gal mixer (1:40, v/v) at 37°C for 2–4 h. X-gal mixer comprises 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₃H₂O, 0.01% sodium deoxycholate, 0.2% Nonidet NP-40, 2 mM MgCl₂ (all from Sigma–Aldrich) in PBS. Next, tissues were rinsed with PBS and mounted in Mowiol (Sigma–Aldrich). Cell nuclei were stained with Nuclear Fast Red (Vector laboratories) according to the manufacturer’s protocol. Using this protocol, we stained cochleae from 3 Lgr4-LacZ knock-out and heterozygous mice. Control experiments found no non-specific staining in cochleae harvested from 3 wild type littermates.

An automated patch-clamp system (Patchliner Octo®, Nanion Technologies, Münich, Germany) was utilized as per standard procedure detailed by the manufacturer and as described previously (Haythornthwaite et al., 2012 (link)). Up to eight cells were measured simultaneously using the Patchliner Octo®, which is equipped with two EPC-10 Quadro patch-clamp amplifiers (HEKA Elektronik, Reutlingen, Germany). The external solution contained 4 mM KCl (Sigma), 140 mM NaCl (Sigma), 2 mM CaCl₂ (Sigma), 1 mM MgCl₂ (Sigma), 10 mM HEPES (Sigma), and 5 mM glucose (Sigma), at pH 7.4. The internal solution (60 mM KF, 50 mM KCl, 20 mM EGTA, 10 mM NaCl, 10 mM HEPES, and 1 mM MgCl₂; Nanion) was supplemented with 3 mM MgCl₂ (Sigma), 0.2 mM GTP (Sigma), and 3 mM ATP (Sigma), adjusted to pH 7.2. A seal-enhancer solution (3 mM KCl, 80 mM NaCl, 35 mM CaCl₂, 10 mM MgCl₂, and 10 mM HEPES [pH 7.4]; Nanion) was used and subsequently changed into external solution upon the establishment of the whole-cell configuration. Patch-ControlHT software (Nanion Technologies, version 12.0.1f9) and Patchmaster software (HEKA Elektronik, version 2×90.4 beta) were utilized for data acquisition. Data were recorded at a sampling rate of 20 kHz (at room temperature) and filtered at 5 kHz.