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 pluripote nt murine embryonic stem (ES) cells into neuronal cells. alpha 1 protein wa s expressed in undifferentiated ES (UES) cells and throughout all stages st udied, 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-sensit ive 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, incl uding cell soma and processes, without evidence of polarization, beta 1 pro tein was expressed in uninduced ES, embryonic bodies (EB) and neuronal cell s. While proteins of the beta 2 and beta 3 isoforms were not detected by im munoblots (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 c ells (beta 3). Message for the beta 2 isoform, however, was not present in neuronal cells, gamma subunit mRNA and protein were undetectable at any sta ge. These results provide further characterization of neuron-like cells obt ained by induction of ES cells in vitro, and establish a model for the expr ession of isoforms of the Na,K-ATPase during neuronal differentiation. The relation to other aspects of neuronal cell development and relevance to a s pecialised function for the alpha 3 subunit in neurons are discussed. (C) 2 000 Elsevier Science B.V, All rights reserved.