Quantifying Ca2+ Current and Permeability in ATP-gated P2X7 Receptors

• Published in: The Journal of biological chemistry 2015-03, Vol.290 (12), p.7930-7942
• Main Authors: Liang, Xin, Samways, Damien S.K., Wolf, Kyle, Bowles, Elizabeth A., Richards, Jennifer P., Bruno, Jonathan, Dutertre, Sébastien, DiPaolo, Richard J., Egan, Terrance M.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - physiology; Animals; Calcium Channels - metabolism; Calcium Transport; Cells, Cultured; Fractional Calcium Current; Humans; Ion Channel; Ligand-gated Ion Channel; Lymphocyte; Macrophage; Macrophages, Peritoneal - cytology; Macrophages, Peritoneal - metabolism; Mice; Molecular Biophysics; Permeability; Pore Dilation; Purinergic Receptor; Receptors, Purinergic P2X7 - physiology; Relative Calcium Permeability
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.627810

ATP-gated P2X7 receptors are prominently expressed in inflammatory cells and play a key role in the immune response. A major consequence of receptor activation is the regulated influx of Ca2+ through the self-contained cation non-selective channel. Although the physiological importance of the resulting rise in intracellular Ca2+ is universally acknowledged, the biophysics of the Ca2+ flux responsible for the effects are poorly understood, largely because traditional methods of measuring Ca2+ permeability are difficult to apply to P2X7 receptors. Here we use an alternative approach, called dye-overload patch-clamp photometry, to quantify the agonist-gated Ca2+ flux of recombinant P2X7 receptors of dog, guinea pig, human, monkey, mouse, rat, and zebrafish. We find that the magnitude of the Ca2+ component of the ATP-gated current depends on the species of origin, the splice variant, and the concentration of the purinergic agonist. We also measured a significant contribution of Ca2+ to the agonist-gated current of the native P2X7Rs of mouse and human immune cells. Our results provide cross-species quantitative measures of the Ca2+ current of the P2X7 receptor for the first time, and suggest that the cytoplasmic N terminus plays a meaningful role in regulating the flow of Ca2+ through the channel. Background: Ca2+ triggers many of the actions of extracellular ATP. Results: We measured the Ca2+ component of the ATP-gated non-selective cation current of P2X7 receptors. Conclusion: Ca2+ flux varied with the species of origin, the splice variant, and the agonist concentration. Significance: Our results suggest that domains that lie outside of the pore regulate the Ca2+ flux of P2X7 receptors.