Glutamate transporter GLAST/EAAT1 directs cell surface expression of FXYD2/γ subunit of Na, K-ATPase in human fetal astrocytes

Na+-dependent uptake of excitatory neurotransmitter glutamate in astrocytes increases cell energy demands primarily due to the elevated ATP consumption by glutamine synthetase and Na+, K+-ATPase. The major pool of GLAST/EAAT1, the only glutamate transporter subtype expressed by human fetal astrocytes in undifferentiated cultures, was restricted to the cytoplasmic compartment. Elevated glutamate concentrations (up to 50 μM) stimulated both glutamate uptake and Na+, K+-ATPase activity and concomitantly increased cell surface expression of GLAST and FXYD2/γ subunit of Na+, K+-ATPase. Intracellular accumulation of glutamate or its metabolites per se was not responsible for these changes since metabolically inert transport substrate, d-aspartate, exerted the same effect. Nanomolar concentrations of TFB-TBOA, a novel nontransportable inhibitor of glutamate carriers, almost completely reversed the action of glutamate or d-aspartate. In the same conditions (i.e. block of glutamate transport) monensin, a potent Na+ ionophore, had no significant effect neither on the activation of Na+, K+-ATPase nor on the cell surface expression of γ subunit or GLAST. In order to elucidate the roles of γ subunit in the glutamate uptake-dependent trafficking events or the activation of the astroglial sodium pump, in some cultures γ subunit/FXYD2 was effectively knocked down using siRNA silencing. Unlike the blocking effect of TFB-TBOA, the down-regulation of γ subunit had no effect neither on the trafficking nor activity of GLAST. However, the loss of γ subunit effectively abolished the glutamate uptake-dependent activation of Na+, K+-ATPase. Following withdrawal of siRNA from cultures, the expression levels of γ subunit and the sensitivity of Na+, K+-ATPase to glutamate/aspartate uptake have been concurrently restored. Thus, the activity of GLAST directs FXYD2 protein/γ subunit to the cell surface, that, in turn, leads to the activation of the astroglial sodium pump, presumably due to the modulatory effect of γ subunit on the kinetic parameters of catalytic α subunit(s) of Na+, K+-ATPase.