Analysis of regulational mechanism of Na pump isoform activities using cells transfected neuron-type isoform.

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Na pump isoforms, "common-type (alpha)" and "neuron-type (alpha2 and alpha3)" isoforms, were expressed in cultured rat cercbral neurons. When the cells were excited with glutamate or maintained in energy-deficient states, the K^+ uptake activities of Na pump isoforms were regulated differently : that of neuron-type isoform increased but that of alpha isoform decreased. To analyze mechanism(s) of the differential regulation of K^+ uptake activities of the isoforms in the neurons, we used T3-3-3 cells which were established by transfecing alpha3 subunit cDNA into BALB/c 3T3 cells and expressed both preexisting alpha1 isoform and transfected alpha3 isoform, In the IC uptake activity of the cells, affinity of a3 isoform for intracellular Na^+ was lower than that of alpha1 isoform. Patch-clamp technique demonstrated that pump current of alpha3 isoform was activated by membrane depolarization but that of alpha1 isoform was not. These findings suggest that the differential regulation of K^+ uptake activities of the isoforms after excitation with glutamate results from the difference in pump kinetics between the isoforms. However, the inhibition of the K^+ uptake activity of alpha1 isoform after excitation occurred in mature neurons, in contrast activation of the activity occurred in immature neurons. Therefore, some mechanism(s) in intracellular signal transduction may also be involved in the differential regulation and developed during maturation in culture. To analyze the mechanism, the T3-3-3 cells were treated with dibutyryl cAMP or forskolin or PMA, however, no or less effect on the isoform activities was detected. Affinity of alpha3 isoform for ATP was smaller than that of alpha1 isoform in the ATPase activity of the T3-3-3 cells. Therefore, it is speculated that a part of the regulation of the Na pump isoform activities in energy-deficient states of the neurons is due to differences in affinities for ATP between the isoforms.