Microcystins (MCs) are a class of cyclic heptapeptides with more than 100 variants produced by cyanobacteria present in surface waters.

Studies employing a single protein nanopore as a molecular recognition element in the development of analytical devices - biosensors, spectrometers, DNA sequencing - have increased considerably in the last decade. Several studies show the potential of these bionanostructures for future stochastic biosensing technology. Stochastic biosensing is an approach that relies on the observation of individual binding events between analyte molecules and a single receptor. This approach is inherent to the organisms that use a single protein nanopore as a key element to start, manage and maintain the chemical and biophysical processes of living cells. Here, we discuss alpha-toxin as a bacterial exotoxin and prototype of a protein nanopore in real-time detection and characterization of molecules in aqueous systems. ]]>

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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% CO 2 . For these experiments, cells were maintained in DMEM supplemented with 10% FBS, 1mM L-glutamine and 50 units/ml Penicillin and 50 mg/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 MU. Solutions used were composed in mM: Pipette (KCl 130; NaCl 10; EGTA 5; Hepes 10) and external (NaCl 140; KCl 54; CaCl 2 2.0; MgCl 2 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 þþ -activated K þ channel (IKCa) and rectifier K þ 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.