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• 영문명: Measurement of Cellular ATP Efflux by Establishment of Biosensor Cells
• 발행기관: 대한약학회
• 저자명: 손민정(Min-Jeong Son) 레안위(Qui Ahn Le) 김준철(Joon-Chul Kim) 김경희(Kyoung-Hee Kim) 우선희(Sun-Hee Woo)
• 간행물 정보: 『약학회지』제63권 제1호(2019년), 30~36쪽, 전체 7쪽
• 주제분류: 의약학 > 기타의약학
• 파일형태: PDF
• 발행일자: 2019.01.30

영문 초록

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.

목차

서 론(Introduction)

실험방법(Experimental Methods)

결과 및 고찰(Results and Discussion)

결 론(Conclusion)

감사의 말씀(Acknowledgment)

References

키워드

ATP-sensing cell P2X7 overexpressing HEK293 cells reporter patch real-time ATP release