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손민정,레안위,김경희,우선희 忠南大學校 醫藥品開發硏究所 2019 藥學論文集 Vol.34 No.-
In this study, we established a biosensor cell that can directly respond to ATP in order to measure ATP release from a single cell in real-time with high time resolution. We made HEK293 cells overexpressing P2X7 purinoceptors, the ATP-gated non-selective cation channel, together with green fluorescence proteins (GFPs). Overexpression of P2X7 receptors was confirmed by reverse transcription- polymerase chain reaction, immunoblotting method, and fluorescence microscopy. In addition, the overexpression of P2X7 receptors was functionally confirmed by measuring ATP-induced cation current in these cells using whole-cell patch clamp technique. Application of ATP-containing external solutions produced inward currents at –70 mV in P2X7-expressing HEK293 cells, in a concentration- dependent manner with an EC50 of 31.2 μM. Maximal P2X7 inward current (14.2 ± 1.76 pA/pF at –70 mV; n=10) was observed at about 0.8 mM ATP. ATP-dependent HEK293 cell currents was almost completely blocked by suramin (30 μM), the P2 purinergic antagonist (0.17 ± 0.13 pA/pF at –70 mV, n=10, P < 0.0001), and they were negligible in the HEK293 cells expressing GFP only (0.28 ± 0.07 pA/pF at –70 mV, n=11). The ATP-biosensor cells may be used to directly quantify ATP released from neighboring cell in real-time, which will minimize both unstirred layer effect and lack of time resolution that normally occur during ATP bioluminescence assay using bulk external solutions.
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