Quantification of the alpha(3) subunit of the Na+/K+-ATPase in developing rat cerebellum

Cerebellar Purkinje neurons of rats have been shown to exhibit a progressiv e increase in resting membrane potential as the animals develop postnatally . The magnitude of this increase was equivalent in magnitude to the increas e in the depolarizing action of ouabain, consistent with a role fur the Na/K+-pump in the hyperpolarization. Ouabain binding sites in whole cerebellu m also increased with age. The present study was undertaken to confirm that the increases in ouabain binding and the electrophysiological responses to ouabain were a consequence of increases in the sodium pump and to determin e whether the changes seen at the whole organ level were reflective of chan ges taking place at the cellular level. Using antibodies directed against t he alpha (1), alpha (2), and alpha (3) subunits of the Na+/K+-ATPase, rats between 13 and 19 days of age exhibited a statistically significant increas e in the relative amount of the a, subunit at the level of the whole organ, as determined by Western and slot blot analyses, with no change in the lev els of either the alpha (1) or the alpha (2) subunit Using immunohistochemi stry, the alpha (3) subunit was shown to increase in both the Purkinje cell layer and the white matter during this postnatal time period, while the al pha (1) subunit increased in the granular layer. These results support and extend previous work, which pointed to a role for the electrogenic sodium p ump in the developmental increase in Purkinje cell membrane potential. Furt hermore, the data provide a cellular mechanism underlying the increase in r esting membrane potential, that is, by the specific modulation of the alpha (3) subunit isoform. (C) 2000 Elsevier Science B.V. All rights reserved.