Kinetic properties of Cl- uptake mediated by Na+-dependent K+-2Cl- cotransport in immature rat neocortical neurons
Kinetic properties of Cl- uptake mediated by Na+-dependent K+-2Cl- cotransport in immature rat neocortical neurons
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DOI：
10.1523/jneurosci.5041-06.2007
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发表时间：
2007-08-08
影响因子：
5.3
通讯作者：
Kilb, Werner
中科院分区：
文献类型：
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作者：
Achilles, Katharina;Okabe, Akihito;Kilb, Werner
GABA是成人神经系统中的主要抑制性神经递质，在未成熟神经元中引起去极化膜反应，这对于早期网络活动的产生至关重要。虽然人们普遍认为GABA的去极化作用是由细胞内Cl-浓度升高（[Cl-]（i））引起的，但未成熟神经元中Cl-蓄积的机制仍存在争议。采用膜片钳、显微荧光、免疫组织化学和分子生物学方法，我们研究了未成熟[出生后第0天（P0）至P3]大鼠新皮质Cajal-Retzius（CR）细胞摄取Cl-的机制。短杆菌肽穿孔膜片钳和6-甲氧基-N-乙基喹啉-微量荧光测量显示稳态[Cl-]（i）类似于30 mM，布美他尼或无Na（+）溶液将其降低至接近被动分布的值，表明Na（+）-K（+）-2Cl（-）共转运亚型1（NKCC 1）参与维持升高的[Cl-]（i）。CR细胞中NKCC 1在mRNA和蛋白水平均有表达。为了详细确定NKCC 1对[Cl（-）]（i）稳态的贡献，在人工[Cl-]（i）消耗后分析Cl-摄取速率。活性Cl-摄取相对缓慢（47.2 +/- 5.0 μ M/s），并被布美他尼或无Na+溶液消除。因此，全细胞膜片钳记录显示CR细胞中Cl（-）电导较低。NKCC 1介导的Cl（-）摄取能力低，足以维持兴奋性GABA能膜反应，但仅在低刺激频率下。总之，我们的结果表明，NKCC 1在未成熟大鼠新皮层的CR细胞中是丰富的，并且由该转运蛋白介导的缓慢Cl-摄取足以维持使GABA反应兴奋所需的高[Cl-]（i）。
GABA, the main inhibitory neurotransmitter in the adult nervous system, evokes depolarizing membrane responses in immature neurons, which are crucial for the generation of early network activity. Although it is well accepted that depolarizing GABA actions are caused by an elevated intracellular Cl- concentration ([Cl-](i)), the mechanisms of Cl- accumulation in immature neurons are still a matter of debate. Using patch- clamp, microfluorimetric, immunohistochemical, and molecular biological approaches, we studied the mechanism of Cl- uptake in Cajal-Retzius (CR) cells of immature [postnatal day 0 ( P0) to P3] rat neocortex. Gramicidin-perforated patch-clamp and 6-methoxy-N-ethylquinolinium-microfluorimetric measurements revealed a steady- state [Cl-](i) of similar to 30 mM that was reduced to values close to passive distribution by bumetanide or Na (+)-free solutions, suggesting a participation of Na (+)-K (+)-2Cl(-) cotransport isoform 1 (NKCC1) in maintaining elevated [Cl-](i). Expression of NKCC1 was found in CR cells on the mRNA and protein levels. To determine the contribution of NKCC1 to [Cl (-)](i) homeostasis in detail, Cl- uptake rates were analyzed after artificial [Cl-](i) depletion. Active Cl- uptake was relatively slow (47.2 +/- 5.0 mu M/s) and was abolished by bumetanide or Na+-free solution. Accordingly, whole-cell patch-clamp recordings revealed a low Cl(-)conductance in CR cells. The low capacity of NKCC1-mediated Cl(-)uptake was sufficient to maintain excitatory GABAergic membrane responses, however, only at low stimulation frequencies. In summary, our results demonstrate that NKCC1 is abundant in CR cells of immature rat neocortex and that the slow Cl- uptake mediated by this transporter is sufficient to maintain high [Cl-](i) required to render GABA responses excitatory.