Functional coupling of Sodium pump and KCC2 in respiratory neural network.

Na^+， K^+- atp酶（钠泵）α 2亚基基因（Atp1a2）敲除纯合小鼠（Atp1a2^<-/->）在出生后因缺乏呼吸而立即死亡。采用脑干-脊髓整体制备研究了Atp1a2^<-/->呼吸相关神经元的活性。在胚胎期18.5的Atp1a2^<-/->胎儿中，从第四颈椎腹根（C4）记录的呼吸运动神经元活动是有缺陷的。经Krebs溶液灌注后，Atp1a2^<-/->脑内细胞外GABA含量较高，与野生型相比，Atp1a2^<-/->脑内C4对电刺激的反应恢复较慢。通过光学记录观察到Atp1a2^<-/->的VLM缺乏抑制性神经活动。gramicide穿孔膜片钳记录显示，Atp1a2^<-/->的VLM神经元细胞内Cl^-浓度较高。α 2亚基和神经元特异性K-Cl共转运体KCC2在纯化的野生型胎儿突触膜部分中共免疫沉淀。基于这些结果，我们提出了一个钠泵α 2亚基与KCC2之间功能耦合的模型，该模型排除了细胞solin呼吸中枢神经元中的Cl^-。这些结果表明，alpha2亚基具有独特的功能，这是在同一神经元中表达的α亚基所不能替代的。

Na^+, K^+-ATPase (Sodium pump) alpha2 subunit gene (Atp1a2) knock-out homozygous mice (Atp1a2^<-/->) died immediately after birth resulting from lack of breathing. The respiratory-related neuron activity in Atp1a2^<-/-> was investigated using a brainstem-spinal cord en bloc preparation. The respiratory motoneuron activity recorded from the fourth cervical ventral root (C4) was defective in Atp1a2^<-/-> fetuses of embryonic day 18.5. The C4 response to electrical stimulation of the ventrolateral medulla (VLM) recovered more slowly in Atp1a2^<-/-> than in wild type during superfusion with Krebs' solution, consistent with the high extracellular GABA in brain of Atp1a2^<-/->. Lack of inhibitory neural activities in VLM of Atp1a2^<-/-> was observed by optical recordings. High intracellular Cl^- concentrations in neurons of the VLM of Atp1a2^<-/-> were detected in gramicidin-perforated patch-clamp recordings. The alpha2 subunit and a neuron-specific K-Cl cotransporter KCC2 were coimmunoprecipitated in a purified synaptic membrane fraction of wild-type fetuses. Based on these results, we propose a model for functional coupling between the Sodium pump alpha2 subunit and KCC2, which excludes Cl^- from the cytosolin respiratory center neurons. These results indicate that alpha2 subunit harbor a unique function that is not compensated by alphal subunit which express in the same neurons.