Bettendorff Lucien, Lakaye Bernard, Margineanu Ilca, Grisar Thierry, Wins Pierre
Center for Cellular and Molecular Neurobiology, University of Liège, 17 place Delcour, B-4020 Liège, Belgium.
J Neurochem. 2002 May;81(4):792-801. doi: 10.1046/j.1471-4159.2002.00858.x.
In immature neurones, the steady-state intracellular Cl- concentration Cl- is generally higher than expected for passive distribution, and this is believed to be due to Na(+)-K(+)-2Cl(-) co-transport. Here, we show that N2a neuroblastoma cells, incubated in HEPES-buffered NaCl medium maintain a Cl- around 60 mm, two- to threefold higher than expected for passive distribution at a membrane potential of - 49 mV. When the cells were transferred to a Cl(-) -free medium, Cl- decreased quickly (t(1/2) < 5 min), suggesting a high Cl- permeability. When the intracellular ATP concentration was reduced to less than 1 mm by metabolic inhibitors, the initial rate of (36) Cl- uptake was strongly inhibited (60-65%) while steady-state Cl- decreased to 24 mm, close to the value predicted from the Nernst equilibrium. Moreover, after reduction of ATP and Cl- by rotenone, the subsequent addition of glucose led to a reaccumulation of Cl-, in parallel with ATP recovery. Internal bicarbonate did not affect Cl- pumping, suggesting that Cl-/HCO(3)(-) exchange does not significantly contribute to active transport. Likewise, Na(+) -K(+) -2Cl(-) co-transport also appeared to play a minor role: although mRNA for the NKCC1 form of the co-transporter was detected in N2a cells, neither the initial rate of (36)Cl- uptake nor steady-state Cl- were appreciably decreased by 10 microm bumetanide or replacement of external Na(+) by choline. These results suggest that a highly active ATP-dependent mechanism, distinct from Na(+) -K(+) -2Cl(-) co-transport, is responsible for most of the inward Cl- pumping in N2a cells.
在未成熟神经元中，稳态细胞内氯离子浓度Cl⁻通常高于被动分布预期值，据信这是由于钠钾氯共转运体所致。在此，我们表明，在HEPES缓冲的NaCl培养基中孵育的N2a神经母细胞瘤细胞维持的Cl⁻约为60 mM，比在-49 mV膜电位下被动分布预期值高两到三倍。当细胞转移至无氯培养基时，Cl⁻迅速下降（半衰期<5分钟），表明氯离子通透性高。当通过代谢抑制剂将细胞内ATP浓度降至低于1 mM时，(³⁶)Cl⁻摄取的初始速率受到强烈抑制（60 - 65%），而稳态Cl⁻降至24 mM，接近能斯特平衡预测值。此外，在鱼藤酮降低ATP和Cl⁻后，随后添加葡萄糖导致Cl⁻重新积累，与ATP恢复平行。细胞内碳酸氢根不影响Cl⁻转运，表明Cl⁻/HCO₃⁻交换对主动转运的贡献不显著。同样，钠钾氯共转运体似乎也起次要作用：尽管在N2a细胞中检测到共转运体NKCC1形式的mRNA，但10 μM布美他尼或用胆碱替代细胞外Na⁺均未明显降低(³⁶)Cl⁻摄取的初始速率或稳态Cl⁻。这些结果表明，一种不同于钠钾氯共转运体的高活性ATP依赖机制负责N2a细胞中大部分的内向Cl⁻转运。
