Cell-type specific expression of Na +,K +-ATPase catalytic subunits in cultured neurons and glia: evidence for polarized distribution in neurons

Abstract

Na +,K +-ATPase (the sodium pump) is a family of proteins consisting of catalytic (α) and glycoprotein (β) subunit isoforms which are differentially expressed in excitable tissue. To gain insight into the cell-type distribution of sodium pump protein, we determined the expression pattern of fetal rat telencephalic cultures, of telencephalic cultures depleted of neurons, and of pure astrocyte cultures. Isoform-specific antibodies were used for immunoblotting and immunohistochemistry, with supplemental [ 3H]ouabain binding to assess levels of functional α2/α3 protein. The results show that neurons of mixed telencephalic cultures uniquely express α3 and high levels of α1. The marked similarity in the distribution of microtubule-associated protein-2 and α1 immunocytochemical staining strongly suggests that α1 subunits are enriched in dendrites. Further, highly correlative growth cone-associated protein-43 and α3 staining is consistent with a preferential expression of α3 subunits in axons, which are also characterized by low levels of α1 and no α2 immunoreactivity. Process-bearing glia are intimately associated with neuronal aggregates and express high levels of both α1 and α2 protein, as well as GFAP. Interestingly, polygonal, flat glia not within neuronal aggregates are weakly immunopositive only for α1 and GFAP. Pure astrocytic cultures possess appreciable α1 protein and GFAP, but lack both α2 and α3 immunoreactivity. As predicted by the immunohistochemical findings, [ 3H]ouabain binding was low in pure astrocytic cultures, and much higher in the neuron-enriched mixed cultures. These observations confirm that neurons express all three catalytic isoforms of the sodium pump. They also suggest that specific α-isoforms may be polarized to targeted membrane regions of neurons. Further, glia intimately associated with neurons express α2, bind significant amounts of [ 3H]ouabain, and possess much higher levels of α1 and GFAP compared to glia not near neurons. Thus, neurons may regulate glial sodium pump expression.