Small-scale molecular motions accomplish glutamate uptake in human glutamate transporters

Abstract

Glutamate transporters remove glutamate from the synaptic cleft to maintain efficient synaptic communication between neurons and to prevent glutamate concentrations from reaching neurotoxic levels. Glutamate transporters play an important role in ischemic neuronal death during stroke and have been implicated in epilepsy and amytropic lateral sclerosis. However, the molecular structure and the glutamate-uptake mechanism of these transporters are not well understood. The most recent models of glutamate transporters have three or five subunits, each with eight transmembrane domains, and one or two membrane-inserted loops. Here, using fluorescence resonance energy transfer (FRET) analysis, we have determined the relative position of the extracellular regions of these domains. Our results are consistent with a trimeric glutamate transporter with a large (>45 A) extracellular vestibule. In contrast to other transport proteins, our FRET measurements indicate that there are no large-scale motions in glutamate transporters and that glutamate uptake is accompanied by relatively small motions around the glutamate-binding sites. The large extracellular vestibule and the small-scale conformational changes could contribute to the fast kinetics predicted for glutamate transporters. Furthermore, we show that, despite the multimeric nature of glutamate transporters, the subunits function independently.

Figure 1.

Fluorescence labeling for FRET between residues in EAAT3. A, Residues labeled by the fluorescent probes. B, I-V plots of the glutamate-activated (1 mm) currents for T121C (•), S207C (▴), S45C (⋄), G283C/M60C (+), A430C/V273C (×), and wt EAAT3 (▾). C, Theoretical FRET transfer rate, k_FRET, for a trimeric or pentameric structure with one donor fluorophore (•) and two or four acceptor fluorophores (). D, Fluorescence labeling curve for A430C (•) and V273C (□) labeled with Alexa Fluor 488 C₅-maleimide. Arrows indicate the amount of labeling used in the intrasubunit experiments. E, Fluorescence emission from an oocyte expressing 334C EAAT3 transporters labeled with only donor fluorophores (▪) and donor and acceptor fluorophores (▵). FRET efficiency is estimated as E = 1 - F_DA/F_D (Selvin, 1995). Endogenous fluorescence from an uninjected, labeled oocyte is also shown (•).

Figure 2.

The quenched donor fluorescence is restored by DTT. Emission spectra for an oocyte expressing EAAT3 M60C transporters: unquenched Alexa Fluor 488 before application of TMR-MTS (▪, solid line), quenched Alexa Fluor 488 after application of TMR-MTS (•, dashed line), and dequenched Alexa Fluor 488 after a subsequent application of 10 mm DTT for 10 min (▵, dotted line). DTT reduces the disulfide between cysteines and TMR-MTS but cannot remove the Alexa Fluor 488 C₅-maleimides. DTT restores the donor fluorescence to its original value.

Figure 3.

Conservation of glutamate transporter structure between bacteria and mammals. A, Extracellular view of a trimeric EAAT3 model that is mostly consistent with distances estimated from our FRET measurements (Table 1). Uncertainties exist in the location of the residues relative to the x-y plane, making a more detailed model impossible (see Materials and Methods). Scale bar, 10 Å. B, Extracellular view of the putative bacterial transporter Glt_PH (Yernool et al., 2004). The extracellular positions of the different transmembrane domains are indicated by the circles. C, A strong correlation exists between the intersubunit distances estimated with FRET (EAAT3 distances) and the distances in the crystal structure (Glt_Ph distances) measured between the homologous residues (measured at the α-C atoms) in neighboring subunits. The FRET estimates are, on average, 8 Å longer, most likely attributable to the size of the linker and the fluorescent probes. The distance between L387C residues (circled) deviates considerably from the distance between the homologous residues in the crystal structure. Note that this residue undergoes the largest movement according to our FRET measurements (Table 1) and is directly connected to loop II (Fig. 1 A), which has been shown to undergo several conformational changes during the uptake cycle (Larsson et al., 2004).

Figure 4.

EAAT3 subunits function independently. Labeling curve for 334C with Alexa Fluor 488 maleimide (▪) superimposed on the percentage of decrease in glutamate-activated current caused by the fluorescent label (○), shown for are presentative oocyte (n = 3). Solid lines are the best exponential fit to the data: τ_fluorescence = 1306 ± 182 μm × min; τ_{current decrease} = 1208 ± 62 μm × min. The dashed line is the predicted decrease in current for a concerted cooperative trimeric transporter. The dotted line indicates the predicted decrease in current for a trimeric transporter in which only one glutamate-binding site can be occupied at a time, and anyone of the binding sites can induce the glutamate-activated current through a common pathway. Norm., Normal.

Figure 5.

Molecular models of glutamate transporters. a, A rocker-switch-type model for transport, which alternately exposes the glutamate-binding site to the intracellular and extracellular solutions by rotating the subunits relative each other. b, A channel model for transport with two gates that open alternately, allowing coupled transport. c, EAAT3 model with a large, extracellular vestibule and three short channels, each with two gates. The gates alternately open to the extracellular solution (left) or to the intracellular solution (right). d, Our data indicate that the three pores transport glutamate independently. Large sphere, glutamate; small sphere, Na⁺. For simplicity, only one Na⁺ and one glutamate are shown to be transported for each transport cycle. It was shown previously that, in each cycle, three Na⁺, one H⁺, and one glutamate are cotransported, whereas one K⁺ is countertransported (Zerangue and Kavanaugh, 1996; Levy et al., 1998).