Evidence for change in current-flux coupling of GLT1 at high glutamate concentrations in rat primary forebrain neurons and GLT1a-expressing COS-7 cells.

Glutamate is the major excitatory neurotransmitter of the central nervous system and is toxic to neurons even at low concentrations. GLT1, the rodent analog of human EAAT2, is primarily responsible for glutamate clearance in the cerebrum. GLT1 was thought to be expressed exclusively in astrocytes in the mature brain. Recently, however, GLT1a was demonstrated in excitatory axon terminals where synaptic glutamate concentration rises above 1 mm during excitatory transmission. GLT1 function in neurons with accurate control of both intracellular and extracellular solutions mimicking synaptic concentration gradients has never been studied. Here we characterized the kinetics of coupled glutamate transporter current in whole-cell configuration and [(3)H]-l-glutamate uptake in cultured rat cerebral neurons across the entire range of synaptic glutamate concentrations. In both neurons and GLT1a-transfected COS-7 cells, the kinetics were similar and revealed two specific components: a high-affinity component with glutamate k(D) value around 15 mum and a low-affinity component with k(D) value around 0.2 mm. The specific low-affinity component was discovered as a result of significant deviation of the transporter current from Michaelis-Menten kinetics in the 100-300 mum concentration range. Activation of the specific low-affinity component led to a two-fold decrease in the current/flux ratio, implying a change in the transport coupling. Our data indicate that GLT1 endogenously expressed in cultured rat forebrain neurons displays high and low glutamate affinity uptake components that are different in current-flux coupling ratios. This property is intrinsic to the protein because it was also observed in GLT1a-transfected COS-7 cells.