Stoichiometry of the Glial Glutamate Transporter GLT-1 Expressed Inducibly in a Chinese Hamster Ovary Cell Line Selected for Low Endogenous Na+-Dependent Glutamate Uptake

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Abstract
Glutamate transport across the plasma membrane of neurons and glia is powered by the transmembrane electrochemical gradients for sodium, potassium, and pH, but there is controversy over the number of Na+cotransported with glutamate. The stoichiometry of glutamate transporters is important because it determines a lower limit to the extracellular glutamate concentration, [glu]o, in both normal and pathological conditions. We used whole-cell clamping to study the stoichiometry of the glial transporter GLT-1, the most abundant glutamate transporter in the brain, expressed under control of the Tet-On system in a Chinese hamster ovary (CHO) cell line selected for low endogenous glutamate transport. After the induction of GLT-1 expression with doxycycline, glutamate evoked a Na+-dependent inward current with the voltage dependence and pharmacology of GLT-1 and acidified the cell cytoplasm. Raising [K+]oaround cells clamped with electrodes containing sodium and glutamate evoked an outward reversed uptake current. These responses were reduced by the specific GLT-1 blocker dihydrokainate (DHK). DHK evoked an outward current with NO3−, but not with Cl−, as the main intracellular anion, suggesting that the anion conductance of the transporter is active even without external glutamate but generates little current in the absence of highly permeable anions like NO3−. Measuring the reversal potential of the transporter current in various ionic conditions suggested that the transport of one glutamate anion is coupled to the cotransport of three Na+and one H+and to the countertransport of one K+. This suggests that in ischemia, when [K+]orises to 60 mm, the reversal of glutamate transporters will raise [glu]oto >50 μm.