Analysis of localization and regulation of Na, K-pump isoforms in neurons.

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Three molecular forms of alpha (alpha1, alpha2, and alpha3) subunits of the Na, K-pump isoforms were identified in cultured granule cells, prepared from 7-day old rat cerebella, by Western blot and histochemical analysis. The subunits were shown to localize on surface of soma and neurites of the cells with ankyrin and spectrin by using confocal laser microscopy. The amounts of these subunits were shown to increase during in vitro differentiation by culture. Na, K-ATPase activities of the Na pump isoforms increased correspondingly during culture, and total activity went up to 333 (]SY.+1.[) 32 (nmol/min・mg of protein) after 13 days. The activity was 4.8-fold, higher than basal level. The brain type (alpha2 and alpha3) isoform activity increased 8.9-fold, and the common type (alpha1) only 3.4-fold. By stimulating the differentiated cells with glutamate, the K+ uptake activities of Na pump isoforms were regulated differently and total activity increased about 20%. The brain type isoform activity increased moro than fourfold by the stimulation, but the common type decreased 23%. Therefore, the percentage of the brain type isoform activity increased from 12% to 43% by the stimulation. The regulation of Na, K-Pump activities by glutamate were abolished by calmodulin antagonist W-7 and did not occur in immature neurons. Number of Na, K-pump on surface of the cells estimated by <@D13@>D1H-ouabain binding study did not change by glutamate stimulation. These results suggest that calmodulin may function in the mechanism to regulate the Na, K-pump isoform activities and that the brain type Na, K-pump isofoum in differentiated neuron is important functionally to restore the ionic gradient after excitation.