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KMID : 0370220190630010030
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Yakhak Hoeji
2019 Volume.63 No. 1 p.30 ~ p.36
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Measurement of Cellular ATP Efflux by Establishment of Biosensor Cells
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Son Min-Jeong
Le Qui Ahn
Kim Joon-Chul
Kim Kyeong-Hee
Woo Sun-Hee
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Abstract
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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 cation channel, 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, n = 10) was observed at about 0.8 mM ATP. ATP-dependent HEK293 cell currents were almost completely blocked by suramin (30 μM), the P2 purinergic antagonist (0.17 ± 0.13 pA/pF, n = 10, p < 0.0001), and they were negligible in the HEK293 cells expressing GFP only (0.28 ± 0.07 pA/pF, n = 11). The ATP-biosensor cells successfully detected ATP release from single atrial myocyte under shear stress at millisecond intervals. This ATP detection method may be used to directly quantify real-time ATP release, which may provide improved spatial and temporal information on cellular ATP release.
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KEYWORD
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ATP-sensing cell, P2X7 overexpressing HEK293 cells, reporter patch, real-time ATP release
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