P2X7R-Mediated Ca2+-Independent D-Serine Release via Pannexin-1 of the P2X7R-Pannexin-1 Complex in Astrocytes

d-serine is a coagonist of N-methyl-d-aspartate (NMDA) subtype of glutamate receptor and plays a role in regulating activity-dependent synaptic plasticity. In this study, we examined the mechanism by which extracellular ATP triggers the release of d-serine from astrocytes and discovered a novel Ca2+-independent release mechanism mediated by P2X(7) receptors (P2X(7)R). Using [H-3] d-serine, which was loaded into astrocytes via the neutral amino acid transporter 2 (ASCT2), we observed that ATP and a potent P2X(7)R agonist, 2(3)-O-(4-benzoylbenzoyl)adenosine-5-triphosphate (BzATP), stimulated [H-3]D-serine release and that were abolished by P2X(7)R selective antagonists and by shRNAs, whereas enhanced by removal of intracellular or extracellular Ca2+. The P2X(7)R-mediated d-serine release was inhibited by pannexin-1 antagonists, such as carbenoxolone (CBX), probenecid (PBN), and (10)Panx-1 peptide, and shRNAs, and stimulation of P2X(7)R induced P2X(7)R-pannexin-1 complex formation. Simply incubating astrocytes in Ca2+/Mg2+-free buffer also induced the complex formation, and that enhanced basal d-serine release through pannexin-1. The P2X(7)R-mediated d-serine release assayed in Ca2+/Mg2+-free buffer was enhanced as well, and that was inhibited by CBX. Treating astrocytes with general protein kinase C (PKC) inhibitors, such as chelerythrine, GF109203X, and staurosporine, but not Ca2+-dependent PKC inhibitor, Go6976, inhibited the P2X(7)R-mediated d-serine release. Thus, we conclude that in astrocytes, P2X(7)R-pannexin-1 complex formation is crucial for P2X(7)R-mediated d-serine release through pannexin-1 hemichannel. The release is Ca2+-independent and regulates by a Ca2+-independent PKC. The activated P2X(7)R per se is also functioned as a permeation channel to release d-serine in part. This P2X(7)R-mediated d-serine release represents an important mechanism for activity-dependent neuron-glia interaction. GLIA 2015;63:877-893