Establishment of an assay for P2X7 receptor-mediated cell death
Song-Yi Lee1, Sooyeon Jo, Ga Eun Lee
1Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
Abstract:
The P2X7 receptor, an ATP-gated cation channel, induces cell death in immune cells and is involved in neurodegenerative diseases. Although the receptor plays various roles in these diseases, the cellular mechanisms involved are poorly understood and antagonists are limited. Here, the development of a cell-based assay for human P2X7 receptor is reported. We established permanent lines of HEK 293 cells expressing a high level of hP2X7 receptor. Functional activity of the hP2X7 receptor was confirmed by whole-cell patch recording of ATP-induced ion currents. Prolonged exposure to ATP resulted in death of the hP2X7-expressing HEK 293 cells and this cell death could be quantified. Two known P2X7 antagonists, PPADS and KN-62, blocked ATP-induced death in a concentration-dependent manner. Thus, this assay can be used to screen for new antagonists of hP2X7 receptors.
Insights
A new cell-based assay for the human P2X7 receptor (hP2X7) was developed. This assay quantifies cell death and can screen for novel P2X7 receptor antagonists, aiding research into neurodegenerative diseases.
Area of Science:
- Pharmacology
- Neuroscience
- Cell Biology
Background:
- The P2X7 receptor, an ATP-gated cation channel, is implicated in immune cell death and neurodegenerative diseases.
- Understanding P2X7 receptor's cellular mechanisms and developing effective antagonists remain significant challenges.
Purpose of the Study:
- To develop a robust cell-based assay for quantifying human P2X7 receptor (hP2X7) activity.
- To establish a screening platform for identifying novel hP2X7 receptor antagonists.
Main Methods:
- Established stable HEK 293 cell lines expressing high levels of hP2X7.
- Utilized whole-cell patch clamp recording to confirm functional ATP-induced ion currents.
- Quantified ATP-induced cell death in hP2X7-expressing cells.
Main Results:
- Demonstrated that prolonged ATP exposure causes quantifiable cell death in hP2X7-expressing HEK 293 cells.
- Confirmed that known P2X7 antagonists (PPADS, KN-62) inhibit ATP-induced cell death in a concentration-dependent manner.
- Validated the assay's utility for screening potential hP2X7 antagonists.
Conclusions:
- The developed cell-based assay provides a reliable method for studying hP2X7 receptor function.
- This assay is suitable for high-throughput screening of new therapeutic compounds targeting the P2X7 receptor.
- Facilitates further investigation into P2X7 receptor's role in disease and drug development.
