Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons.
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 Ca(2+) ([Ca(2+)]i) in nearly 35% of the KCl-responsive cells. These Ca(2+) responses disappeared in a Ca(2+)-free medium supplemented with EGTA. Mn(2+) quench experiments showed that OAG activated Ca(2+)-conducting channels that were also permeant to Ba(2+). The OAG-induced Ca(2+) responses were unaffected by nifedipine or omega-conotoxin GVIA (Sigma-Aldrich, Saint-Quentin Fallavier, France) but blocked by 1-[beta-(3-(4-Methoxyphenyl)propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF)-96365 and Gd(3+). Replacing Na(+) ions with N-methyl-D-glucamine diminished the amplitude of the OAG-induced Ca(2+) responses showing that the Ca(2+) 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 [Ca(2+)]i but not the protein kinase C activator phorbol 12-myristate 13-acetate. Moreover, the OAG-induced Ca(2+) 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 [Ca(2+)]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 Ca(2+)-permeable channels displaying TRPC6-like properties. This is the first report revealing the existence of second messenger-operated channels in cortical neurons.
Tu
Peng
P
CNRS UMR 5249, Grenoble, France.
Kunert-Keil
Christiane
C
Lucke
Silke
S
Brinkmeier
Heinrich
H
Bouron
Alexandre
A
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D001643:Bridged Bicyclo Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D003512:Cyclohexanones / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology*; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects*; D018408:Patch-Clamp Techniques; D010696:Phloroglucinol / Q000031:analogs & derivatives / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D015290:Second Messenger Systems / Q000187:drug effects*; D012964:Sodium / Q000378:metabolism; D050052:TRPC Cation Channels / Q000502:physiology*; D013729:Terpenes / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism
C520323:1-oleoyl-2-acetoyl-sn-glycerol; D000814:Aniline Compounds; D001643:Bridged Bicyclo Compounds; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D003512:Cyclohexanones; D004075:Diglycerides; D004791:Enzyme Inhibitors; C409648:Fluo 4; D050052:TRPC Cation Channels; D013729:Terpenes; D014966:Xanthenes; D011189:Potassium Chloride; C036535:1,6-bis(cyclohexyloximinocarbonyl)hexane; D012964:Sodium; D010696:Phloroglucinol; C001654:hyperforin; D002118:Calcium; D016257:Fura-2