Figure 5

Neferine activates ryanodine receptor in turn to mobilize cytosolic calcium level. (A) Neferine induced calcium dynamic change in HeLa. HeLa cells stained with FLIPR Calcium 6 Assay Kit were treated with 10 and 20 μM of neferine, then immediately subjected to calcium dynamic measurement by FLIPR Tetra High-Throughput Cellular Screening System. Data from the chart represents mean values ± S.D. of three independent experiments. (B) Single-cell imaging of Neferine-mediated mobilization of cytosolic calcium. HeLa cells seeded in 35 mm confocal disc were incubated with 5 μM of Fluo 3/AM in HBSS buffer at 37 °C for 30 min. Cells were then washed with HEPES buffer saline and incubated at 37 °C for another 10 min. Calcium signal was monitored by Applied Precision DeltaVision Elite in real-time mode for consecutive 5 min during the addition of 10 μM neferine in HBSS buffer. Chart represents the mean intensity of fluorescence signal at 523 nm. (C) Computation docking of SERCA with neferine. The SERCA protein was represented as ribbon diagram. Key residues around the binding pocket are shown as lines and the neferine is presented as sticks. The hydrogen bonds were labeled as red dashed lines. Extra precision (XP) scoring for thapsigargin and neferine are −7.23 and −7.32 respectively. (D) Inhibition of Ca²⁺ ATPase (SERCA) activity in skeletal muscle SR by neferine. Experiments were measured at 25 °C (pH 7.2) using the coupled enzyme assay as described in¹⁰. (E) Ca²⁺ releasing effect of neferine on inositol 1,4,5-trisphosphate (InsP₃) receptor. The InsP₃ receptor rich rat brain microsomes were pre-incubated with or without neferine (20 or 40 μM), and then subjected to IP₃ (20 μM) treatment. The calcium release was immediately measured within 5 min. (F) Time-dependent Ca²⁺ release from skeletal muscle SR upon addition of neferine. The ryanodine receptor rich skeletal muscle SR was pre-incubated with or without RyR inhibitor, tetracaine (1 mM) for 1 min and then subjected to neferine (20μM) treatment. The calcium release was immediately measured within 5 min. (G) Dose-dependent Ca²⁺ release from skeletal muscle SR upon addition of neferine. The ryanodine receptor rich skeletal muscle SR was treated with indicated concentrations of neferine in the presence or absence of RyR inhibitor, ruthenium red (40 μM) or tetracaine (1 mM). The calcium release was immediately measured by Fluo-3 (free acid). All experimental points are the mean ± SD of 3 determinations.