Tu Peng, Kunert-Keil Christiane, Lucke Silke, Brinkmeier Heinrich, Bouron Alexandre
CNRS UMR 5249, Grenoble, France.
J Neurochem. 2009 Jan;108(1):126-38. 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 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.
脂质二酰甘油（DAG）类似物1-油酰基-2-乙酰基-sn-甘油（OAG）被用于验证从E13小鼠胚胎解离的皮质神经元中DAG敏感通道的存在。钙成像实验表明，OAG使近35%的对KCl有反应的细胞内Ca(2+)（[Ca(2+)]i）浓度升高。在添加了EGTA的无钙培养基中，这些Ca(2+)反应消失。Mn(2+)淬灭实验表明，OAG激活了对Ba(2+)也有通透性的Ca(2+)传导通道。OAG诱导的Ca(2+)反应不受硝苯地平或ω-芋螺毒素GVIA（西格玛奥德里奇公司，法国圣康坦法拉维耶）的影响，但被1-[β-(3-(4-甲氧基苯基)丙氧基)-4-甲氧基苯乙基]-1H-咪唑盐酸盐（SKF）-96365和Gd(3+)阻断。用N-甲基-D-葡萄糖胺取代Na(+)离子会降低OAG诱导的Ca(2+)反应的幅度，表明Ca(2+)内流是通过Na(+)依赖和非Na(+)依赖机制介导的。用荧光Na(+)指示剂CoroNa Green进行的实验表明，OAG升高了[Na(+)]i。与OAG一样，DAG脂肪酶抑制剂RHC80267增加了[Ca(2+)]i，但蛋白激酶C激活剂佛波酯12-肉豆蔻酸酯13-乙酸酯没有增加。此外，OAG诱导的Ca(2+)反应不受蛋白激酶C激活或抑制的调节，但氟芬那酸增强了这种反应，氟芬那酸可增加通过C型瞬时受体电位蛋白家族（TRPC）6通道的电流。此外，应用TRPC6通道的特异性激活剂贯叶连翘素可升高[Ca(2+)]i。全细胞膜片钳记录表明，贯叶连翘素激活了非选择性阳离子通道。它们被SKF-96365阻断，但被氟芬那酸增强。总之，我们的数据表明存在对贯叶连翘素和OAG敏感的Ca(2+)通透通道，具有类似TRPC6的特性。这是首次报道揭示皮质神经元中存在第二信使操作通道。
