title: Subcellular distribution and early signalling events of P2X7 receptors from mouse cerebellar granule neurons.
abstract: The subcellular distribution and early signalling events of P2X7 receptors were studied in mouse cerebellar granule neurons. Whole-cell patch-clamp recordings evidenced inwardly directed non-desensitizing currents following adenosine 5'-triphosphate (ATP; 600 µM) or 2'-3'-o-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP; 100 µM) administration to cells bathed in a medium with no-added divalent cations (Ca(2+) and Mg(2+)). Nucleotide-activated currents were inhibited by superfusion of 2.5 mM Ca(2+), 1.2 mM Mg(2+) or 100 nM Brilliant Blue G (BBG), hence indicating the expression of ionotropic P2X7 receptors. Fura-2 calcium imaging showed [Ca(2+)]i elevations in response to ATP or BzATP at the somas and at a small number of axodendritic regions of granule neurons. Differential sensitivity of these [Ca(2+)]i increases to three different P2X7 receptor antagonists (100 nM BBG, 10 μM 4-[(2S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester, KN-62, and 1 μM 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine hydrochloride hydrate, A-438079) revealed that P2X7 receptors are co-expressed with different P2Y receptors along the plasmalemma of granule neurons. Finally, experiments with the fluorescent dye YO-PRO-1 indicated that prolonged stimulation of P2X7 receptors does not lead to the opening of a membrane pore permeable to large cations. Altogether, our results emphasise the expression of functional P2X7 receptors at both the axodendritic and somatic levels in mouse cerebellar granule neurons, and favour the notion that P2X7 receptors might function in a subcellular localisation-specific manner: presynaptically, by controlling glutamate release, and on the cell somas, by supporting granule neuron survival against glutamate excytotoxicity.
authors.affiliation: Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain; Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain; Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense de Madrid, 28040 Madrid, Spain. Electronic address: sanchezn@ucm.es.
authors.forename: Jesús
authors.initials: J
authors.lastname: Sánchez-Nogueiro
authors.affiliation: Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain; Departamento de Medicina Preventiva, Salud Pública e Inmunología Médica y Microbiología. Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, 28922 Madrid, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Marín-García
authors.affiliation: Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain; Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Diego
authors.initials: D
authors.lastname: Bustillo
authors.affiliation: Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain; Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Luis Alcides
authors.initials: LA
authors.lastname: Olivos-Oré
authors.affiliation: Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain; Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: María Teresa
authors.initials: MT
authors.lastname: Miras-Portugal
authors.affiliation: Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain; Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Antonio R
authors.initials: AR
authors.lastname: Artalejo
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism*; D016257:Fura-2 / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D013777:Tetrazoles / Q000494:pharmacology
chemical_list: C523668:3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methylpyridine; D058920:Purinergic P2X Receptor Antagonists; D011725:Pyridines; D058486:Receptors, Purinergic P2X7; D013777:Tetrazoles; D018698:Glutamic Acid; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2
keywords: ATP; BzATP; Cerebellar granule neurons; Electrophysiology; Microfluorometry; P2X7 receptors