What Type of Ion Channels are Identified on Osteosarcoma Cells Membrane

Human Osteosarcoma (SaOs-2) cell line is a good and recognized model for in vitro calcification. These cells present osteoblast-like properties and can be mineralized under appropriate conditions. There is not a full electrophysiological characterization on these cells. Our goal was to screen ion channels on SaOs-2 cells membrane. SaOs-2 cells were obtained from tissue bank at UFRJ (BCRJ # 0217) and maintained in an incubator at 37°C with 5% CO2. For these experiments, cells were maintained in DMEM supplemented with 10% FBS, 1mM L-glutamine and 50 units/ml Penicillin and 50 μg/ml Streptomycin. Cells were detached from culture flasks using trypsin-EDTA. After centrifugation at 1.000 rpm for 2 minutes, cells were recovered in culture medium and plated into a small single plate and allowed to attach for at least 1 hour. Electrophysiological recording was performed in the whole-cell configuration using a patch clamp amplifier in voltage clamp mode. The signal was registered in 5 KHz. Micropipette tip resistance 3-5 MΩ. Solutions used were composed in mM: Pipette (KCl 130; NaCl 10; EGTA 5; Hepes 10) and external (NaCl 140; KCl 54; CaCl2 2.0; MgCl2 0.5; Glucose 10; Hepes 10). Tetraethylammonium (TEA), Iberotoxin (IBTX) and Tetrodotoxin (TTX) (10 mM, 100 nM and 100mM, respectively) were used as inhibitors for Ca++-activated K+ channel (IKCa) and rectifier K+ current channels (IKDR). Slow activation current similar to IKDR and another one as fast activation current similar to IKCa channel were observed. Both currents (IKCa; IKDR) were decreased approximately 65 % (n=3) by TEA when compared to relative control condition. IKCa was decreased approximately 86 % (n=3) to IBTX specifically. The inward current was entirely abolished by TTX application. Further experiments are still under investigation to determine more channels on SaOs-2 cells.