Mag fura 2

Fluorescence of 150 nM Mag-Fura-2 (ThermoFisher Scientific) (Ex/Em 340/510 nm) saturated with Zn(II) was measured on a CLARIOstar Plus spectrophotometer (BMG Labtech) at 25°C in response to increasing concentrations of metal-free protein. Five technical replicates of each sample were analyzed in a black half-volume 384-well microtiter plate (Greiner Bio One). All experiments were performed in Chelex-100 treated 50mM MOPS (pH 7.2) 200 mM NaCl buffer. Fluorescence values were analyzed using a one-site non-linear fit model in GraphPad Prism (Version 8) using the experimentally-derived K_D of Mag-Fura-2 (47 nM) to determine the K_Dfor Zn(II) binding by AdcAII.

Astragaloside IV (purity at 98%) was purchased from Shanghai Bogoo Biotechnology Company (Shanghai Co. Ltd., China). Palmitic acid, BAPTA-AM and SKF96365 were obtained from Sigma–Aldrich (St. Louis, MO, USA). Mag-Fura-2, Rhod-2 and JC-1 were purchased from Invitrogen (Carlsbad, CA, USA). Fluo-4 was purchased from Dojindo (Kumamoto, Japan) and OAG was purchased from MedChem Express (Monmouth Junction, NJ, USA).

All the flash experiments were performed under the normal saline in bath contained the following (in mM): 140 NaCl, 5 KCl, 1.8 CaCl₂, 2 MgCl₂, 10 HEPES and 10 Glucose with the PH adjusted to 7.4. Pipette solution contained the following (in mM): 120 KCl, 10 NP-EGTA, 8.05 CaCl₂, 2 K₂ATP, 0.4 Fura-4f and 0.4 Mag-Fura-2 and 10 HEPES with the pH adjusted to 7.2. For the experiments of BK current activated by calcium influx, the extracellular solution contained the following (in mM): 150 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES (pH 7.38); Pipette solution contained the following (in mM): 140 KCl, 10 NaCl, 5 EGTA, 1 MgCl2, 10 HEPES (pH 7.36). For the experiments of BK kinetic modeling, intracellular solutions with different free Ca²⁺ were made by mixing (in mM) 160 MeSO3K and 10 HEPES with 5 EGTA (for 0 μM Ca²⁺); 5 EGTA and 3.25 CaCl2 (for 1 μM Ca²⁺); 5 HEDTA and 2.99 CaCl2 (for 10 μM Ca²⁺); 0.1 CaCl2 (for 100 μM Ca²⁺); 0.3 CaCl2 (for 300 μM Ca²⁺), with the pH adjusted to 7.0. Free Ca²⁺ was estimated by the EGTAETC program (E. McCleskey, Vollum Institute, Portland, OR). Pipette solution contained the following (in mM): 160 MeSO₃K, 2 MgCl₂, 10 HEPES with the pH adjusted to 7.0. All the chemicals were attained from Sigma except that NP-EGTA, Mag-Fura-2 and Fura-4f were from Invitrogen.