Analysis of mechanisms regulation ion transport activities of Na pump isoforms in neurons.

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Three Na pump isoforms α1, α2 and α3 isoforms, are present in cultured neurons and ion transport activities of the isoforms are differentially regulated. Here we show that the inhibition of α2/α3, a neuron type Na pump, isoform by extracellular K^+ is a key mechanism of the differential regulation. Only the α2/α3 isoform is inhibited by physiological concentrations of K^+ under basal conditions in mature neurons. The inhibition was vanished after glutamate excitation of the neurons and by incubation with a calmodulin antagonist or an inhibitor of CaM kinase II. In contrast to mature neurons the inhibition is small in immature neurons, in which the isoform activities are not regulated differentially. Additionally, when ATP concentration in the neurons becomes low, the inhibition diminishes and the differential regulation becomes increasingly prominent. We also show that astrocyte-derived factors instruct mouse ES cells to differentiate into neurons. Cultured in astrocyte-conditioned med … More ium (ACM) under free-floating conditions, within 4 days, colonies of undifferentiated ES cells give rise to floating spheres with a concentric stratiform structure. Specifically, the spheres have a periphery of neural stem cells, a core of dividing ES cells and an intermediate layer. Culturing the spheres on an adhesive substrate in ACM promotes neurogenesis. The attached spheres give rise to clusters of cells containing neurons at the periphery, and many neurons with neurites migrate from the clusters within 5 days. Gene expression analyses and electrophysiology confirm the neuronal properties of the cells. The neurons express a neuron type Na pump, the α2 and α3 isoforms. Immunofluorescence analyses show that many NF^+ neurons and neurites are also positive for tyrosine hydroxylase. In contrast, neither astrocytes nor oligodendrocytes are formed. This pathway provides a new insight into mechanisms of neurogenesis and also provides a simple procedure for generation of neural stem cells and neurons. Less