Gegelashvili, M; Rodriguez-Kern, A; Sung, L; Shimamoto, K; Gegelashvili, G
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 50Â [mu]M) stimulated both glutamate uptake and Na+, K+-ATPase activity and concomitantly increased cell surface expression of GLAST and FXYD2/[gamma] 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 [gamma] subunit or GLAST. In order to elucidate the roles of [gamma] subunit in the glutamate uptake-dependent trafficking events or the activation of the astroglial sodium pump, in some cultures [gamma] subunit/FXYD2 was effectively knocked down using siRNA silencing. Unlike the blocking effect of TFB-TBOA, the down-regulation of [gamma] subunit had no effect neither on the trafficking nor activity of GLAST. However, the loss of [gamma] subunit effectively abolished the glutamate uptake-dependent activation of Na+, K+-ATPase. Following withdrawal of siRNA from cultures, the expression levels of [gamma] subunit and the sensitivity of Na+, K+-ATPase to glutamate/aspartate uptake have been concurrently restored. Thus, the activity of GLAST directs FXYD2 protein/[gamma] subunit to the cell surface, that, in turn, leads to the activation of the astroglial sodium pump, presumably due to the modulatory effect of [gamma] subunit on the kinetic parameters of catalytic [alpha] subunit(s) of Na+, K+-ATPase.