Expression, activity and distribution of Na,K-ATPase subunits during in vitro neuronal induction

The expression pattern of the alpha and beta isoforms and the gamma subunit of the Na,K-ATPase was investigated during in vitro induction of pluripotent murine embryonic stem (ES) cells into neuronal cells. alpha 1 protein was expressed in undifferentiated ES (UES) cells and throughout all stages studied, in contrast, alpha 3 protein was prominent only when neuronal cells have reached full differentiation. In this model, neuron-depleted cultures did not express the alpha 3 isoform, indicating its specificity for mature neuronal cells. UES possessed Na,K-ATPase activity consistent with a single isoform (alpha 1), whereas in fully mature neuronal cells a ouabain-sensitive isoform (alpha 3) accounted for 27+/-4% of the activity, and a ouabain-resistant isoform (alpha 1) 66+/-3%. Immunocytochemistry of mature neuronal cells for alpha 1 and alpha 3 proteins showed a similar distribution, including cell soma and processes, without evidence of polarization, beta 1 protein was expressed in uninduced ES, embryonic bodies (EB) and neuronal cells. While proteins of the beta 2 and beta 3 isoforms were not detected by immunoblots (except for beta 2 in UES), their mRNAs were detected in UES and EB (beta 2 and beta 3), and in immature and fully differentiated neuronal cells (beta 3). Message for the beta 2 isoform, however, was not present in neuronal cells, gamma subunit mRNA and protein were undetectable at any stage. These results provide further characterization of neuron-like cells obtained by induction of ES cells in vitro, and establish a model for the expression of isoforms of the Na,K-ATPase during neuronal differentiation. The relation to other aspects of neuronal cell development and relevance to a specialised function for the alpha 3 subunit in neurons are discussed. (C) 2000 Elsevier Science B.V, All rights reserved.