Malfunction of Respiratory-Related Neuronal Activity in Na+, K+-ATPase α2 Subunit-Deficient Mice Is Attributable to Abnormal Cl-Homeostasis in Brainstem Neurons

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Abstract
Na+, K+-ATPase α2 subunit gene (Atp1a2) knock-out homozygous mice (Atp1a2-/-) died immediately after birth resulting from lack of breathing. The respiratory-related neuron activity inAtp1a2-/-was investigated using a brainstem-spinal corden blocpreparation. The respiratory motoneuron activity recorded from the fourth cervical ventral root (C4) was defective inAtp1a2-/-fetuses of embryonic day 18.5. The C4 response to electrical stimulation of the ventrolateral medulla (VLM) recovered more slowly inAtp1a2-/-than in wild type during superfusion with Krebs' solution, consistent with the high extracellular GABA in brain ofAtp1a2-/-. Lack of inhibitory neural activities in VLM ofAtp1a2-/-was observed by optical recordings. High intracellular Cl-concentrations in neurons of the VLM ofAtp1a2-/-were detected in gramicidin-perforated patch-clamp recordings. The α2 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 Na+, K+-ATPase α2 subunit and KCC2, which excludes Cl-from the cytosol in respiratory center neurons.