ATP-감지 세포 제작 및 그 기능성 검증 = Establishment of ATP-Sensing Cells and Confirmation of Their Functionality

다국어 초록 (Multilingual 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 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.