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Measurement of Cellular ATP Efflux by Establishment of Biosensor Cells
Yakhak Hoeji 2019;63(1):30-36
Published online February 28, 2019
© 2019 The Pharmaceutical Society of Korea.

Min-Jeong Son, Qui Ahn Le, Joon-Chul Kim, Kyoung-Hee Kim, and Sun-Hee Woo#

College of Pharmacy, Chungnam National University
Correspondence to: Sun-Hee Woo, College of Pharmacy, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, South Korea, Tel: +82-42-821-5924, Fax: +82-42-823-6566, E-mail: shwoo@cnu.ac.kr
Received January 15, 2019; Revised January 20, 2019; Accepted February 1, 2019.
Abstract
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.
Keywords : ATP-sensing cell, P2X7 overexpressing HEK293 cells, reporter patch, real-time ATP release


February 2019, 63 (1)
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