Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons


Address correspondence and reprint requests to Alexandre Bouron, Laboratoire de Chimie et Biologie des Métaux, UMR CNRS 5249, CEA, 17 rue des Martyrs, 38054 Grenoble, France. E-mail: alexandre.bouron@cea.fr


The lipid diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was used to verify the existence of DAG-sensitive channels in cortical neurons dissociated from E13 mouse embryos. Calcium imaging experiments showed that OAG increased the cytosolic concentration of Ca2+ ([Ca2+]i) in nearly 35% of the KCl-responsive cells. These Ca2+ responses disappeared in a Ca2+-free medium supplemented with EGTA. Mn2+ quench experiments showed that OAG activated Ca2+-conducting channels that were also permeant to Ba2+. The OAG-induced Ca2+ responses were unaffected by nifedipine or omega-conotoxin GVIA (Sigma-Aldrich, Saint-Quentin Fallavier, France) but blocked by 1-[β-(3-(4-Methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF)-96365 and Gd3+. Replacing Na+ ions with N-methyl-d-glucamine diminished the amplitude of the OAG-induced Ca2+ responses showing that the Ca2+ entry was mediated via Na+-dependent and Na+-independent mechanisms. Experiments carried out with the fluorescent Na+ indicator CoroNa Green showed that OAG elevated [Na+]i. Like OAG, the DAG lipase inhibitor RHC80267 increased [Ca2+]i but not the protein kinase C activator phorbol 12-myristate 13-acetate. Moreover, the OAG-induced Ca2+ responses were not regulated by protein kinase C activation or inhibition but they were augmented by flufenamic acid which increases currents through C-type transient receptor potential protein family (TRPC) 6 channels. In addition, application of hyperforin, a specific activator of TRPC6 channels, elevated [Ca2+]i. Whole-cell patch-clamp recordings showed that hyperforin activated non-selective cation channels. They were blocked by SKF-96365 but potentiated by flufenamic acid. Altogether, our data show the presence of hyperforin- and OAG-sensitive Ca2+-permeable channels displaying TRPC6-like properties. This is the first report revealing the existence of second messenger-operated channels in cortical neurons.