The glutamate and chloride permeation pathways are colocalized in individual neuronal glutamate transporter subunits

Abstract

Glutamate transporters have a homotrimeric subunit structure with a large central water-filled cavity that extends partially into the plane of the lipid bilayer (Yernool et al., 2004). In addition to uptake of glutamate, the transporters also mediate a chloride conductance that is increased in the presence of substrate. Whether the chloride channel is located in the central pore of the trimer or within the individual subunits has been controversial. We find that coexpression of wild-type neuronal glutamate transporter EAAT3 subunits with subunits mutated at R447, a residue governing substrate selectivity (Bendahan et al., 2000), results in transport activity consistent with two distinct noninteracting populations of transporters, in agreement with previous work suggesting that each subunit operates independently to transport substrate (Awes et al., 2004; Grewer et al., 2005; Koch and Larsson, 2005). In wild-type homotrimeric transporters, the glutamate concentration dependence of the anion conductance and the kinetics of glutamate flux were isolated and measured, and the anion channel activation was fitted to analytical expressions corresponding to (1) a central pore gated by binding to one or more subunits and (2) a channel pore in each subunit. The data indicate that glutamate-binding sites, transport pathways, and chloride channels reside in individual subunits in a trimer and function independently.

Figure 1.

hEAAT3 structural homology model based on the P. horikoshii sodium-dependent glutamate transporter homolog structure (Yernool et al., 2004). A, Trimeric structure comprising three identical subunits. B, A single subunit showing a possible orientation of bound l-Glu. The R447 residue that was mutated in this study is shown in black. Homology models were constructed using SYBYL (Tripos, St. Louis, MO) and PyMOL (DeLano Scientific, Palo Alto, CA).

Figure 2.

Selectivity of currents and transport mediated by homotrimeric and heterotrimeric wild-type (WT) and R447C mutant subunits. A, The l-Glu concentration response of transporter currents for homomultimeric WT (▵; EC₅₀ = 85.7 μm ± 19; n_Hill = 0.84 ± 0.02; n = 7), R447C (▴; n = 3), and heteromultimeric R447C+WT (○; EC₅₀ = 70.4 ± 12.5 μm; n_Hill = 0.89 ± 0.07; n = 6) subunits. The extracellular solution contained Cl–Ringer with V_m = −20 mV. B, l-Ala concentration response of transporter currents for the homomultimeric WT (▴; n = 4), R447C (▵; EC₅₀ = 30.8 ± 9 μm; n_Hill = 0.85 ± 0.05; n = 4), and heteromultimeric R447C coexpressed with WT (○; EC₅₀ = 20.8 ± 6 μm; n_Hill = 0.91 ± 0.05; n = 6) subunits. The extracellular solutions contained NO₃⁻—Ringer with V_m = 20 mV. All currents were fitted to I/I_max = Sⁿ/(Sⁿ + EC₅₀ⁿ). Heterotrimer experiments for A and B were performed in the same oocytes. C, Radiolabel uptake of 30 μm [³H]d-Asp or l-Ala verifies selectivity switch caused by mutation of R447. Error bars indicate SEM.

Figure 3.

Analytical modeling of anion channel gating. Shown are models depicting anion channel schemes for the following: (1) independent gating in each subunit of the trimer (black line; EC₅₀ = 45 μm; n_Hill = 1), (2) central pore gated by noncooperative binding to one or more subunits (red line; EC₅₀ = 11.90; n_Hill = 1.48), (3) central pore gated by occupancy of two or more subunits (blue line; EC₅₀ = 46.33; n_Hill = 1.57), and (4) central pore gated by occupancy of three subunits (green line; EC₅₀ = 180.50; n_Hill = 1.18).

Figure 4.

Modeling of channel activation data. A, Concentration dependencies of isolated coupled transport current (●; EC₅₀ = 43.7 μm; n = 8; Cl⁻–Ringer, −20 mV) and the uncoupled anion current (○; EC₅₀ = 42.8; n = 8; NO₃–Ringer, −20 mV). The coupled and anion currents were fitted to (I/I_max = [l-Glu]/[l-Glu] + EC₅₀). B, The model predictions and the experimental anion conductance data are shown for an anion channel within each subunit (black line; χ² = 8.08), one central anion pore gated by noncooperative binding to one or more subunits (red line; χ² = 1369), one central anion pore gated by noncooperative binding to at least two subunits (blue line; χ² = 202), or one central anion pore gated by noncooperative binding of all three subunits (green line; χ² = 867). The measured coupled transport constant used for the fitting was 44 μm. Error bars indicate SEM.