Both reentrant loops of the sodium-coupled glutamate transporters contain molecular determinants of cation selectivity

Abstract

In the brain, glutamate transporters terminate excitatory neurotransmission by removing this neurotransmitter from the synapse via cotransport with three sodium ions into the surrounding cells. Structural studies have identified the binding sites of the three sodium ions in glutamate transporters. The residue side-chains directly interact with the sodium ions at the Na1 and Na3 sites and are fully conserved from archaeal to eukaryotic glutamate transporters. The Na2 site is formed by three main-chain oxygens on the extracellular reentrant hairpin loop HP2 and one on transmembrane helix 7. A glycine residue on HP2 is located closely to the three main-chain oxygens in all glutamate transporters, except for the astroglial transporter GLT-1, which has a serine residue at that position. Unlike for WT GLT-1, substitution of the serine residue to glycine enables sustained glutamate transport also when sodium is replaced by lithium. Here, using functional and simulation studies, we studied the role of this serine/glycine switch on cation selectivity of substrate transport. Our results indicate that the side-chain oxygen of the serine residues can form a hydrogen bond with a main-chain oxygen on transmembrane helix 7. This leads to an expansion of the Na2 site such that water can participate in sodium coordination at Na2. Furthermore, we found other molecular determinants of cation selectivity on the nearby HP1 loop. We conclude that subtle changes in the composition of the two reentrant hairpin loops determine the cation specificity of acidic amino acid transport by glutamate transporters.

Keywords: amino acid transport; computational biology; electrogenic process; glutamate; ion selectivity; membrane transporter reconstitution; molecular dynamics simulations; neurotransmitter transport; reentry; site-directed mutagenesis.

Figure 1.

Structure of Glt_Ph highlighting the sodium sites and the studied positions. A, the transport domain (orange), the HP1 and HP2 loops (pink), and the trimerization domain (blue) are highlighted in protomer A of the Glt_Ph trimer. Protomers B and C are shown in transparent rendering. B, zoom into the binding site of protomer A, highlighting the 3 sodium ions as well as residue 279 on the HP1 loop, residue 354 on the HP2 loop, and residue 307 on TM7. C visualizes the residue conservation, color coded from red (not conserved) to dark blue (completely conserved) as determined by the Consurf analysis.

Figure 2.

Characterization of transport activity and cation selectivity of polar Ser-440 mutants of GLT-1. HeLa cells expressing GLT-1–WT and hydrophilic replacement mutants of Ser-440 in the pBluescript SK(−) background were measured for sodium and lithium-dependent d-[³H]aspartate uptake at room temperature for 10 min, as described under “Experimental procedures.” The data are given as percentage of activity of sodium-dependent WT GLT-1 uptake (A) and percentage of transport in the presence of lithium to that in the presence of sodium (B). Individual data points are shown as triangles, the horizontal line represents the mean ± S.D. (error bars) of at least three separate experiments performed in triplicate. Stars represent significance of p < 0.0001.

Figure 3.

Characterization of transport activity and cation selectivity of nonpolar Ser-440 mutants of GLT-1. HeLa cells expressing GLT-1 and nonpolar Ser-440 mutants in the pBluescript SK(−) background were measured for sodium (A)- and lithium (B)-dependent d-[³H]aspartate uptake (plotted as % Li/Na) at room temperature for 10 min, as described under “Experimental procedures.” Individual data points are shown as triangles, the horizontal line represents the mean ± S.D. (error bars) of at least three separate experiments performed in triplicate. Stars represent significance of p < 0.0001.

Figure 4.

Cell-surface biotinylation of GLT-1 Ser-440 mutants. HeLa cells transfected with GLT-1 (WT), the indicated mutants or with the vector alone (SK), were biotinylated and processed as described under “Experimental procedures.” The indicated markers were run in the lane to the left of WT and contain Prestained Protein Marker, Broad Range (catalogue number P7706S) was from New England Biolabs. The gel shown is representative of three different experiments. The intensities of all bands were summed up, because they are transporter specific (see the SK control lane of the empty vector).

Figure 5.

Characterization of d-[³H]aspartate and l-[³H]aspartate transport activity and cation selectivity of Glt_Ph-WT, G354S, and G354L. The transporters were purified, reconstituted, and assayed for transport as described under “Experimental procedures.” The medium inside of the liposomes contained 0.12 m KP_i, pH 7.4. The external medium contained 150 mm NaCl or LiCl supplemented with valinomycin (2.5 μm). Nigericin (5 μm) was added for background determination. Transport was measured at room temperature for 30 min. Results are expressed as percent of sodium-driven WT Glt_Ph uptake (A) and percentage of lithium to sodium activity (B). Individual data points are shown as upward and inverted triangles, representing uptake with d-[³H]aspartate and l-[³H]aspartate, respectively. The horizontal line represents the mean ± S.D. (error bars) of at least three separate experiments performed in triplicate. Stars represent significance of p < 0.0001.

Figure 6.

Probability distributions of distances. A, the distance between the Cα atoms of residue 279 on HP1 and residue 354 on HP2, averaged over all simulations, quantifies HP2 gate closure. The geometry of the Na2 site and therefore of sodium binding is quantified by measuring the probability distribution of distances across the Na2 sites between carbonyl oxygens of Thr-308 on TM7 and Ile-350 (B) and Ile-352 (C) on HP2 loop for the protomers that maintained a bound sodium ion.

Figure 7.

Na2 ion-binding site. Representative final structures show the Na2 site as observed in (A) the WT Glt_Ph simulations, (B) the G354S mutant, and (C) the G354L mutant. Residue Ala-307/Gly-354, and the backbone of HP2 and TM7 in proximity of the Na2 site are highlighted. The residues Thr-308, Ile-350, and Thr-352 that coordinate the sodium ion are indicated. The range of the corresponding observed distances is shown in Fig. 6. The hydrogen bond between the hydroxyl group of G354S and the backbone carbonyl oxygen of Ala-307 is highlighted in B. Once formed, the hydrogen bond is stable throughout the simulations. Water molecules coordinating the sodium ion are shown, if within 0.4 nm from the sodium ion.

Figure 8.

Characterization of d-[³H]aspartate and l-[³H]aspartate transport activity and cation selectivity of EAAC1-WT and G410S. HeLa cells expressing EAAC1-WT and G410S in the pBluescript SK(−) background were assayed for sodium (A)- and lithium (B)-dependent d- and l-[³H]aspartate uptake (plotted as % Li/Na) at room temperature for 10 min, as described under “Experimental procedures.” Individual data points are shown as upward and inverted triangles representing uptake with d-[³H]aspartate and l-[³H]aspartate, respectively. The horizontal line represents the mean ± S.D. (error bars) of at least three separate experiments performed in triplicate. Stars represent significance of p < 0.0001.

Figure 9.

Characterization of transport activity and cation selectivity of EAAC1-WT and HP1 mutants. HeLa cells expressing EAAC1-WT, HP1 single and double mutants in the pBluescript SK(−) background were measured for sodium (A)- and lithium (B)-dependent d-[³H]aspartate uptake (plotted as % Li/Na) at room temperature for 10 min, as described under “Experimental procedures.” Individual data points are shown as triangles and the horizontal line represents the mean ± S.D. (error bars) of at least three separate experiments performed in triplicate. Stars represent significance of p < 0.0001.

Figure 10.

Characterization of transport activity and cation selectivity of GLT-1 HP1 mutant T360I in GLT-1–WT and S440G background. HeLa cells expressing GLT-1–WT and the indicated mutants in the pBluescript SK(−) background were measured for sodium (A)- and lithium (B)-dependent d-[³H]aspartate uptake (plotted as % Li/Na) at room temperature for 10 min, as described under “Experimental procedures.” Individual data points are shown as triangles and the horizontal line represents the mean ± S.D. (error bars) of at least three separate experiments performed in triplicate. One star represents significance of p < 0.01 and two stars represents significance of p < 0.0001.