Glutamate release and synapsin-I phosphorylation induced by P2X 7 receptors activation in cerebellar granule neurons David Leo ´n, Jesu ´s Sa ´nchez-Nogueiro, Patricia Marı ´n-Garcı ´a, M a Teresa Miras-Portugal * Department of Biochemistry, Veterinary Faculty, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain Received 20 August 2007; received in revised form 30 November 2007; accepted 7 December 2007 Available online 15 December 2007 Abstract The present work reports that activation of P2X 7 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 EC 50 value of 1.95 mM. The involvement of P2X 7 receptor was suggested when maximal secretory response was significantly reduced by the selective P2X 7 antagonist Brilliant Blue G (BBG; 100 nM) and abolished by removing extracellular Ca 2+ . The involvement of P2X 7 receptor on synaptic vesicle release was confirmed by measuring the release of FM 1-43 dye. In this case, pharmacological activation of P2X 7 was achieved with the more selective agonist 2 0 -3 0 -o-(4-benzoylbenzoyl)-adenosine 5 0 -triphosphate (BzATP; 100 mM) showing a significant FM 1-43 release that was blocked by BBG (100 nM), by Zn 2+ ions (100 mM), both P2X 7 blockers, but not by suramin (100 mM), antagonist of P2X 1 , P2X 2 , P2X 3 and P2X 5 . In addition, BzATP, through P2X 7 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 mM) and KN-93 (10 mM). These results suggest, therefore, that Ca 2+ entrance mediated by P2X 7 receptor induces glutamate release and in parallel synapsin-I phosphorylation. # 2007 Elsevier Ltd. All rights reserved. Keywords: ATP; P2X; CaMKII; Synapsin-I; Glutamate release In the brain, nucleotides are involved in the regulation of several physiological and patophysiological processes by binding ligand-gated cation channels (P2X) or metabotropic (P2Y) receptors (Burnstock, 2005; North and Verkhratsky, 2006). In the last few years numerous evidences point that ATP, in addition to postsynaptic effects, can also initiate or control the release of neurotransmitter by binding presynaptic P2X receptors. Thus, by using different experimental approaches, such as synaptosomes or brain slices obtained from several regions of CNS, various authors have shown the involvement of presynaptic P2X receptors in the modulation of different neurotransmitter release including glutamate, GABA and acetylcholine (Rodrigues et al., 2005; Ireland et al., 2004; Papp et al., 2004; Gualix et al., 2003; Sperla ´gh et al., 2002; Gomez-Villafuertes et al., 2001). However, the detailed sequence of events and the molecular components through which P2X modulate synaptic transmission remain unknown. Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is likely candidate for such intermediates. This multifunctional enzyme at presynaptic level has been implicated in the regulation of neurotransmitter release by phosphorylating synapsin-I protein. This effect is due in large part to the ability of the synapsins to regulate the availability of synaptic vesicles for release. Thus, under resting conditions synapsin-I tethers synaptic vesicles to each other and to cytoskeletal elements inhibiting neurotransmitter release. Once phosphorylated, synapsin-I dissociate from synaptic vesicles and, as a consequence, vesicles can mobilize and fuse with the plasma membrane (Leenders and Sheng, 2005). We recently showed the abundant presence of P2X 7 receptors in presynaptic regions from cultured granule neurons (Hervas et al., 2003). These receptors, once activated, produced a significant increase in CaMKII phosphorylation mostly in fibers, which correspond with P2X 7 subdistribution (Leo ´ n et al., 2006). In the present work we show that the activation of P2X 7 www.elsevier.com/locate/neuint Available online at www.sciencedirect.com Neurochemistry International 52 (2008) 1148–1159 * Corresponding author. Tel.: +34 91 394 3894; fax: +34 91 394 3909. E-mail address: mtmiras@vet.ucm.es (M.T. Miras-Portugal). 0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.12.004