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

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+]o around 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+]o rises to 60 mM, the reversal of glutamate transporters will raise [glu]o to >50 microM.

Fig. 1.

Voltage dependence of glutamate-evoked currents in CHO cells expressing GLT-1. A, Currents evoked by 100 μm glutamate (black bars) in a cell clamped to the potentials shown by each trace. B, Peak glutamate-evoked current as a function of voltage for the cell inA; similar results were obtained from seven cells.

Fig. 2.

Glutamate dependence of uptake currents mediated by GLT-1. A, Current record from a cell superfused with solutions containing different glutamate concentrations (top line). B, Average dose–response curve for the uptake current in five cells. The smooth curve is a best-fit Michaelis–Menten curve with the parameters shown in theboxed inset.

Fig. 3.

Properties of forward and reversed uptake mediated by GLT-1 in CHO cells. A, Sodium dependence: removing external Na⁺ (replaced with choline) abolished the current evoked by glutamate (GLU; bars) at −67 mV.B, Sensitivity to dihydrokainate (DHK) with solutions containing Cl⁻ as the main anion. After a control response to 300 μm glutamate, 200 μm DHK was found to evoke no current change. Applying glutamate in DHK evoked a current that was smaller than in control solution (note that the reduction produced by DHK is less than that quoted in the text for 200 μm glutamate because [glutamate] was 300 μm here). Applying glutamate again after the DHK was washed out evoked a response similar in size to the initial control.C, Responses to 200 μm DHK and 200 μm glutamate, using a pipette solution containing 130 mm NO₃⁻. Left panels, DHK evoked an outward current that was smaller at more positive potentials, i.e., a conductance decrease. Right panels, Glutamate evoked an inward current that was larger at more negative potentials, i.e., a conductance increase.D, Voltage dependence of responses obtained as inC (from a different cell; similar results were obtained in three cells). E, F, Reversed uptake, alone (E) or superimposed on an inward current through K+ channels (F), evoked by raising [K⁺]_o from 0 to 60 mmaround cells clamped with a pipette containing 10 mmNa-glutamate. E, Raising [K⁺]_o evoked an outward current (at +40 mV) that was suppressed by 200 μm external glutamate.F, In another cell, raising [K⁺]_o evoked an inward current (at 0 mV) that was increased by 200 μm external glutamate. Subtracting the current in the presence from that in the absence of glutamate revealed the outward K⁺-evoked reversed uptake current component (ΔI). The return of the current changes to baseline is not shown, because the duration of the [K⁺]_o elevation that was applied was different in the presence and absence of glutamate.

Fig. 4.

Changes of pH generated by GLT-1. For each panel the top trace is the membrane current, and thebottom trace is the BCECF fluorescence (F), quantified as the fractional change ΔF/F. In vitrocalibration (Rink et al., 1982) indicates that a ΔF/F of 0.05 corresponds to a pH change of 0.05 units. A, Change of fluorescence of BCECF (excited at 490 and emitted at 530 nm) and membrane current evoked by 50 μm glutamate in a cell clamped to −63 mV. When glutamate evokes a step change of uptake current, a change in the slope of the fluorescence record is seen (as expected if a proton influx accompanying uptake is proportional to the uptake current).B, Comparison of BCECF fluorescence and membrane current (at −63 mV) changes that are seen when excitation is at 440 and 490 nm during the application of d-aspartate (50 μm). d-Aspartate always evokes an inward current but produces no fluorescence change with 440 nm excitation. Thesmall break in the current trace for 490 nm shows where an electrical artifact was removed. C,d-Aspartate evokes a pH change in a cell clamped to −20 mV, i.e., above the reversal potential for H⁺ (−41 mV), as well as at more negative potentials. For this experiment 1 mm amiloride was present in the superfusion solution.

Fig. 5.

Reversal potential of the transporter and its substrate dependence. A, Specimen data showing the current changes produced by dihydrokainate (DHK; 200 μm) at various potentials (shown by eachtrace) in control solution (containing 101 mm Na⁺, 42.5 mmK⁺, and 100 μm glutamate, pH 7.4). At negative potentials the transporter moves glutamate and net positive charge in the inward direction, and DHK produces an outward current. At positive potentials the transporter runs in the other direction, and DHK produces an inward current. The amplitudes of current changes like this are plotted with the opposite sign in B–D to show the current that is blocked by DHK. B–D, Shifts of reversal potential of the current suppressed by DHK, produced by altering the external concentrations of Na⁺, H⁺, K⁺, and glutamate.Straight lines are linear regression fits to the data.B, Voltage dependence of the transporter current in control solution with 101 mm[Na⁺]_o (filled circles), then in solution with reduced [Na⁺]_o (open circles), and then again in control solution (filled triangles). C, Similar data but for a reduction of [H⁺]_o (pH_o = 8.0).D, I–V relation for the DHK-suppressed transporter current in solution with reduced [K⁺]_o (10 mm; open circles), then in control solution (42.5 mm[K⁺]_o; filled circles), and then in 10 mm[K⁺]_o again (open triangles). E, Data as in B but for an increase of external glutamate concentration from the control value (100 μm) to 300 μm. Specimen data are shown for single cells (rather than averaged over all cells) for each solution change because of small variations in the initial reversal potential in each cell (quantified in Table 1), presumably reflecting small differences in intracellular [Na⁺], [H⁺], [K⁺], or [Glu⁻], which would add noise to the shift of theI–V relation. The theoretical predictions (Eqs. 2-5) for the reversal potential shifts are independent of the exact intracellular substrate concentrations and are compared with mean data (averaged over all cells) in Table 1.