Glutamate release and synapsin-I phosphorylation induced by P2X7 receptors activation in cerebellar granule neurons

The present work reports that activation of P2X7 receptor induces synaptic vesicle release in granule neurons and phosphorylation of synapsin-I by calcium–calmodulin-dependent protein kinase II (CaMKII), which in turn modulates secretory event. ATP, in absence of magnesium, induced a concentration-dependent glutamate release with an EC50 value of 1.95 μM. The involvement of P2X7 receptor was suggested when maximal secretory response was significantly reduced by the selective P2X7 antagonist Brilliant Blue G (BBG; 100 nM) and abolished by removing extracellular Ca2+. The involvement of P2X7 receptor on synaptic vesicle release was confirmed by measuring the release of FM 1-43 dye. In this case, pharmacological activation of P2X7 was achieved with the more selective agonist 2′-3′-o-(4-benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP; 100 μM) showing a significant FM 1-43 release that was blocked by BBG (100 nM), by Zn2+ ions (100 μM), both P2X7 blockers, but not by suramin (100 μM), antagonist of P2X1, P2X2, P2X3 and P2X5. In addition, BzATP, through P2X7 receptor activation, significantly increased the phosphorylation of synapsin-I, the main presynaptic target of CaMKII. Both effects mediated by BzATP were inhibited by the CaMKII inhibitors KN-62 (10 μM) and KN-93 (10 μM). These results suggest, therefore, that Ca2+ entrance mediated by P2X7 receptor induces glutamate release and in parallel synapsin-I phosphorylation.