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

Abstract The expression pattern of the α and β isoforms and the γ subunit of the Na,K-ATPase was investigated during in vitro induction of pluripotent murine embryonic stem (ES) cells into neuronal cells. α1 protein was expressed in undifferentiated ES (UES) cells and throughout all stages studied. In contrast, α3 protein was prominent only when neuronal cells have reached full differentiation. In this model, neuron-depleted cultures did not express the α3 isoform, indicating its specificity for mature neuronal cells. UES possessed Na,K-ATPase activity consistent with a single isoform (α1), whereas in fully mature neuronal cells a ouabain-sensitive isoform (α3) accounted for 27±4% of the activity, and a ouabain-resistant isoform (α1) 66±3%. Immunocytochemistry of mature neuronal cells for α1 and α3 proteins showed a similar distribution, including cell soma and processes, without evidence of polarization. β1 protein was expressed in uninduced ES, embryonic bodies (EB) and neuronal cells. While proteins of the β2 and β3 isoforms were not detected by immunoblots (except for β2 in UES), their mRNAs were detected in UES and EB (β2 and β3), and in immature and fully differentiated neuronal cells (β3). Message for the β2 isoform, however, was not present in neuronal cells. γ 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 α3 subunit in neurons are discussed.