The glutamate-activated anion conductance in excitatory amino acid transporters is gated independently by the individual subunits

Abstract

Excitatory amino acid transporters (EAATs) use sodium and potassium gradients to remove glutamate from the synapse and surrounding extracellular space, thereby sustaining efficient synaptic transmission and maintaining extracellular glutamate concentrations at subneurotoxic levels. In addition to sodium-driven glutamate uptake, EAATs also mediate a glutamate-activated chloride conductance via a channel-like mechanism. EAATs are trimeric proteins and are thought to comprise three identical subunits. Previous studies have shown that the sodium-driven uptake of glutamate occurs independently in each of the three subunits. In contrast, a recent study reports high Hill coefficients for the activation of EAAT anion currents by glutamate and suggests that the subunits function cooperatively in gating the chloride conductance. In the present work, we find that the Hill coefficient for the activation of the anion current by glutamate is approximately 1 in both EAAT3 and EAAT4. Furthermore, we also used fluorescent labeling and inactivation correlation on EAAT3 and EAAT4 to determine whether the glutamate-activated chloride conductance is gated independently or cooperatively by the transporters. We found that both glutamate uptake currents and glutamate-activated chloride currents are mediated independently by each subunit of an EAAT multimer. It has been suggested that EAAT subtypes with particularly large anion conductances can directly influence the excitability of presynaptic terminals in certain neurons. Thus, the finding that the anion conductance is gated independently, rather than cooperatively, is important because it significantly alters predictions of the influence that EAAT-mediated anion currents will have on synaptic transmission at low glutamate concentrations.

Figure 1.

Glutamate-induced currents in EAAT3 and EAAT4. Representative current–voltage relationships for 1 mm glutamate application to oocytes expressing EAAT3 (A) and EAAT4 (B). ●, Current recorded in NaSCN–Ringer's solution (SCN⁻); ▴, current from cells dialyzed in gluconate–Ringer's solution for 24 h and recorded in gluconate–Ringer's solution (gluc⁻). C, Dose–response for glutamate currents in EAAT3 recorded in NaSCN–Ringer's solution at +60 mV; K_m = 122.2 ± 15 μm, n_H = 0.91 ± 0.10 (n = 3). D, Dose–response for glutamate currents in EAAT4 recorded in NaSCN–Ringer's solution at +60 mV; K_m = 2.62 ± 0.55 μm, n_H = 0.91 ± 0.13 (n = 3). Insets in C, D, Expanded view of experimental data fit with the Hill equation (solid curves) versus simulated data fit with the Hill equation with identical K_m and I/I_{max glu} as in experimental data but with n_H = 3 (dashed curves).

Figure 2.

FLIC in EAAT3 S334C. A, Fluorescence-labeling curve for EAAT3 S334C with TMR-MTS in NaCl–Ringer's solution (○) superimposed on the percentage decrease in current activated by 1 mm glutamate (glu) and recorded in NaCl–Ringer's solution at −80 mV (■) or −30 mV (▴). Dashed line indicates the predicted decrease in current for a concerted cooperative trimeric transporter (see Materials and Methods, Fluorescence measurements). For fluorescence labeling, τ = 38.7 ± 5.4 μm × min; for current decrease at −80 mV, τ = 36.1 ± 3.8 μm × min; for current decrease at −30 mV, τ = 33.2 ± 3.0 μm × min. B, Fluorescence-labeling curve for EAAT3 S334C with TMR-MTS in NaSCN–Ringer's solution (○) superimposed on the percentage decrease in current activated by 1 mm glutamate and recorded in NaSCN–Ringer's solution at +60 mV (■). For fluorescence labeling, τ = 47.7 ± 3.6 μm × min.; for current decrease at +60 mV, τ = 49.9 ± 2.7 μm × min. C, Fluorescence-labeling curve for EAAT3 S334C with TMR-MTS in NaCl–Ringer's solution (○) superimposed on the percentage decrease in current activated by 30 μm glutamate and recorded in NaCl–Ringer's solution at −80 mV (■) or −30 mV (▴). For fluorescence labeling, τ = 38.6 ± 5.5 μm × min; for current decrease at −80 mV, τ = 38.0 ± 3.7 μm × min; for current decrease at −30 mV, τ = 36.9 ± 3.9 μm × min. D, Fluorescence-labeling curve for EAAT3 S334C with TMR-MTS in NaSCN–Ringer's solution (○) superimposed on the percentage decrease in current activated by 30 μm glutamate and recorded in NaSCN–Ringer's solution at +60 mV (■). For fluorescence labeling, τ = 56.0 ± 4.8 μm × min; for current decrease at +60 mV, τ = 56.3 ± 6.2 μm × min. In all experiments, the fluorescence from uninjected oocytes exposed to identical labeling conditions as in the experimental was subtracted as background. All data are representative for n = 3 experiments.

Figure 3.

FLIC in EAAT4 S388C. Fluorescence-labeling curve for EAAT4 S388C with TMR-MTS in NaSCN–Ringer's solution (○) superimposed on the percentage decrease in current activated by 300 μm glutamate and recorded in NaSCN–Ringer's solution at +60 mV (■). For fluorescence labeling, τ = 36.0 ± 4.0 μm × min; for current decrease at +60 mV, τ = 32.1 ± 3.9 μm × min. Fluorescence from uninjected oocytes was subtracted as background. Data are representative for n = 3 experiments.

Figure 4.

Glutamate-induced currents in HEK-293 cells expressing rat EAAT4. A, Representative current–voltage relationships for the indicated concentrations of glutamate. B, Unsubtracted currents elicited in response to the indicated concentrations of glutamate (0–100 μm). Cells were stepped to a potential of +80 mV from a holding potential of 0 mV. C, Normalized dose–response relationship for glutamate-activated currents at +80 mV (n = 4 cells; means ± SEM). Background currents in the absence of glutamate were subtracted. Data were fit with the Hill equation (solid line) with K_m = 0.73 and n_H = 1.02. For comparison, dashed line represents fit with the Hill equation: K_m = 0.73 and n_H = 3.