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

• Published in: Journal of neurochemistry Vol. 108; no. 1; pp. 126 - 138
• Main Authors: Tu, Peng, Kunert-Keil, Christiane, Lucke, Silke, Brinkmeier, Heinrich, Bouron, Alexandre
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 2009; Blackwell Publishing Ltd; Wiley-Blackwell; Wiley
• Subjects: Adrenals. Interrenals; Adrenomedullary hormones. Regulation; Aniline Compounds; Aniline Compounds - metabolism; Animals; Bicyclo Compounds; Biochemistry; Biological and medical sciences; brain; Bridged Bicyclo Compounds - pharmacology; Calcium; Calcium - metabolism; Calcium Channel Blockers; Calcium Channel Blockers - pharmacology; Calcium Channels; Calcium Channels - physiology; Cell membranes. Ionic channels. Membrane pores; Cell structures and functions; Cells, Cultured; Cerebral Cortex; Cerebral Cortex - cytology; cortex; Cyclohexanones; Cyclohexanones - pharmacology; Developmental biology; Diglycerides; Diglycerides - pharmacology; Embryo, Mammalian; Enzyme Inhibitors; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; Fura-2; Fura-2 - metabolism; Life Sciences; Membrane Potentials; Membrane Potentials - drug effects; Membrane Potentials - physiology; Mice; Mice, Inbred C57BL; Molecular and cellular biology; Neurobiology; Neurology; Neurons; Neurons - drug effects; Neurons and Cognition; Patch-Clamp Techniques; Phloroglucinol; Phloroglucinol - analogs & derivatives; Phloroglucinol - pharmacology; Potassium Chloride; Potassium Chloride - pharmacology; Rodents; Second Messenger Systems; Second Messenger Systems - drug effects; Sodium; Sodium - metabolism; Terpenes; Terpenes - pharmacology; transient receptor potential canonical; TRPC Cation Channels; TRPC Cation Channels - physiology; Vertebrates: endocrinology; Xanthenes; Xanthenes - metabolism; Embryo; Protein kinase C; Transient receptor potential protein; Calcium; transient receptor potential canonical; calcium channels; Central nervous system; Lipids; Activation; Ionic channel; neurons; Encephalon; cortex; Enzyme; Transferases; Rodentia; Ions; brain; Vertebrata; Mammalia; Neuron; TRP ion channel; Sodium; Mouse; Second messenger; Cations
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/j.1471-4159.2008.05752.x

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.