Differential contribution of the Na+-K+-2Cl- cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons
Differential contribution of the Na+-K+-2Cl- cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons
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DOI：
10.1096/fj.08-116012
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发表时间：
2009-04-01
期刊：
影响因子：
4.8
通讯作者：
Callewaert, G.
中科院分区：
文献类型：
--
作者：
Chabwine, J. N.;Talavera, K.;Callewaert, G.
质膜氯 (Cl-) 途径在神经元生理学中发挥着重要作用。在这里，我们研究了 NKCC1 协同转运蛋白（次级主动 Cl- 摄取机制）在培养的大鼠背根神经节神经元 (DRGN) 和源自胎儿期胚胎第 14 天的运动神经元 (MN) 处理 Cl- 中的作用。短杆菌肽穿孔膜片钳记录显示，DRGN 通过布美他尼和 Na+ 敏感机制积累细胞内 Cl-，这表明 NKCC1 的功能表达。 Western blotting证实了NKCC1在DRGN和MN中的表达，但免疫细胞化学实验显示在MN的树突中表达受限，这与DRGN中的均一表达形成对比。在去极化或超极化膜电位的 GABA(A) 受体激活过程中，MN 和 DRGN 都可以很容易地加载或耗尽 Cl-。加载后，两种细胞类型恢复至静息 Cl- 浓度的速率（即 [Cl-](i) 减少）相似，并且不受降低细胞外 Na+ 浓度的影响。相比之下，在对照条件下，DRGN 的耗竭恢复（即 [Cl-](i) 增加）明显更快，但在低细胞外 Na+ 下则不然。实验观察结果可以通过细胞内 Cl- 动力学的数学模型来重现，其中 DRGN 比 MN 表现出更高的 NKCC1 活性和更小的 Cl- 处理体积。基于这些结果，我们得出结论，胚胎 DRGN 表现出比胚胎 MN 更高的 NKCC1 体细胞功能表达。 DRGN 中的高 NKCC1 活性对于维持高 [Cl-](i) 非常重要，而 MN 中较低的 NKCC1 活性则允许神经元活动期间出现较大的 [Cl-](i) 变化。-Chabwine, J. N.、Talavera, K.、Verbert, L.、Eggermont, J.、Vanderwinden, J.-M.、De Smedt, H.、Van Den Bosch, L.、Robberecht, W.，Callewaert，G. Na+-K+-2Cl(-) 协同转运蛋白 NKCC1 对大鼠胚胎背根神经节神经元和运动神经元氯处理的差异贡献。 FASEB J. 23, 1168-1176 (2009)
Plasma membrane chloride (Cl-) pathways play an important role in neuronal physiology. Here, we investigated the role of NKCC1 cotransporters (a secondary active Cl- uptake mechanism) in Cl- handling in cultured rat dorsal root ganglion neurons (DRGNs) and motor neurons (MNs) derived from fetal stage embryonic day 14. Gramicidin-perforated patch-clamp recordings revealed that DRGNs accumulate intracellular Cl- through a bumetanide- and Na+-sensitive mechanism, indicative of the functional expression of NKCC1. Western blotting confirmed the expression of NKCC1 in both DRGNs and MNs, but immunocytochemistry experiments showed a restricted expression in dendrites of MNs, which contrasts with a homogeneous expression in DRGNs. Both MNs and DRGNs could be readily loaded with or depleted of Cl- during GABA(A) receptor activation at depolarizing or hyperpolarizing membrane potentials. After loading, the rate of recovery to the resting Cl- concentration (i.e., [Cl-](i) decrease) was similar in both cell types and was unaffected by lowering the extracellular Na+ concentration. In contrast, the recovery on depletion (i. e., [Cl-](i) increase) was significantly faster in DRGNs in control conditions but not in low extracellular Na+. The experimental observations could be reproduced by a mathematical model for intracellular Cl- kinetics, in which DRGNs show higher NKCC1 activity and smaller Cl--handling volume than MNs. On the basis of these results, we conclude that embryonic DRGNs show a higher somatic functional expression of NKCC1 than embryonic MNs. The high NKCC1 activity in DRGNs is important for maintaining high [Cl-](i), whereas lower NKCC1 activity in MNs allows large [Cl-](i) variations during neuronal activity.-Chabwine, J. N., Talavera, K., Verbert, L., Eggermont, J., Vanderwinden, J.-M., De Smedt, H., Van Den Bosch, L., Robberecht, W., Callewaert, G. Differential contribution of the Na+-K+-2Cl(-) cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons. FASEB J. 23, 1168-1176 (2009)