Cysteine scanning mutagenesis of transmembrane helix 3 of a brain glutamate transporter reveals two conformationally sensitive positions

Abstract

Glutamate transporters in the brain remove the neurotransmitter from the synapse by cotransport with three sodium ions into the surrounding cells. Recent structural work on an archaeal homolog suggests that, during substrate translocation, the transport domain, including the peripheral transmembrane helix 3 (TM3), moves relative to the trimerization domain in an elevator-like process. Moreover, two TM3 residues have been proposed to form part of a transient Na3' site, and another, Tyr-124, appears close to both Na3' and Na1. To obtain independent evidence for the role of TM3 in glutamate transport, each of its 31 amino acid residues from the glial GLT-1 transporter was individually mutated to cysteine. Except for six mutants, substantial transport activity was detected. Aqueous accessibility of the introduced cysteines was probed with membrane-permeant and membrane-impermeant sulfhydryl reagents under a variety of conditions. Transport of six single cysteine mutants, all located on the intracellular side of TM3, was affected by membrane-permeant sulfhydryl reagents. However, only at two positions could ligands modulate the reactivity. A120C reactivity was diminished under conditions expected to favor the outward-facing conformation of the transporter. Sulfhydryl modification of Y124C by 2-aminoethyl methanethiosulfonate, but not by N-ethylmaleimide, was fully protected in the presence of sodium. Our data are consistent with the idea that TM3 moves during transport. Moreover, computational modeling indicated that electrostatic repulsion between the positive charge introduced at position 124 and the sodium ions bound at Na3' and Na1 underlies the protection by sodium.

FIGURE 1.

Transport cycle and location of the putative sodium-binding sites. A, transport cycle. The order of binding of the three sodium ions, l-glutamate, and the proton is not indicated. After l-glutamate (or other substrates such as d- and l-aspartate) and the co-ions bind to the transporter from the external medium (extracellular (EC)) (step 1), they are translocated (step 2) and released (step 3) into the inside of the cell (cytoplasm (Cyt)). Subsequently, potassium binds from the intracellular side (step 4) and, after translocation to the outside (step 5), is released there (step 6). After completion of this second half-cycle, a new translocation cycle can commence. The empty transporter (T) is not capable of mediating the translocation step, as indicated by the disabled arrows. The steps in this scheme are reversible, and therefore, elevated levels of extracellular potassium can cause release of intracellular glutamate. When a bulky non-transportable analog (blocker) binds from the extracellular medium together with sodium (the stoichiometry (n) is unknown), the transporter remains locked in the outward-facing conformation (dashed line) because translocation cannot proceed. B, model of rat GLT-1 in an outward-open conformation. The approximate extents of the lipid bilayer are shown with horizontal lines; the extracellular side is at the top, and the cytoplasm is at the bottom. Only two protomers are shown for clarity. The transporter domain consisting of TM1 (dark blue), TM3 (light blue), TM6 (green), TM7 (wheat), TM8 (magenta), HP1 (yellow), and HP2 (orange) is believed to move in an elevator-like motion relative to the trimer interface domain (gray). C, close-up view of the boxed region in B, showing the ions (blue spheres) in the four proposed ion-binding sites, with the substrate (gray) and key residues shown as sticks.

FIGURE 2.

Transport activity of TM3 cysteine mutants. HeLa cells expressing Cys-less GLT-1 and cysteine mutants of TM3 in the pBluescript SK⁻ background were analyzed for sodium-dependent d-[³H]aspartate uptake at room temperature for 10 min as described under “Experimental Procedures.” The data are given as the percentage of activity of cysteine-less WT GLT-1 and are the mean ± S.E. (error bars) of at least three separate experiments performed in triplicates.

FIGURE 3.

Cell surface biotinylation of single cysteine mutants. HeLa cells transfected with Cys-less WT GLT-1 (CL-GLT-1), the indicated mutants, or the pBluescript SK⁻ vector alone (SK) were subjected to the biotinylation protocol and processed as described under “Experimental Procedures.” Shown are the biotinylated (A) and total (B) samples. The markers shown were run in the lane to the left of the SK lane and contained prestained broad-range protein markers (P7708S, New England Biolabs).

FIGURE 4.

Effects of sulfhydryl reagents on transport activity of TM3 cysteine mutants. HeLa cells transiently expressing each of the indicated TM3 cysteine mutants were preincubated for 5 min with transport solution containing 150 mm NaCl and 20 μm dl-TBOA with or without 1 mm MTSET (A) or 150 mm KCl with or without 1.5 mm NEM (B) as described under “Experimental Procedures,” followed by washing and d-[³H]aspartate transport. Results for each mutant are expressed as a percentage of its untreated control and represent the mean ± S.E. (error bars) of at least three experiments performed in triplicates. The means of the mutants were compared with that of Cys-less WT GLT-1 (CL-GLT-1) using a one-way analysis of variance with Dunnett's post hoc multiple comparison test. *, p < 0.01.

FIGURE 5.

Effects of the composition of the external medium on mutants reactive to NEM. The indicated mutants were transiently expressed in HeLa cells. Subsequently, the cells were preincubated for 5 min with or without 1 mm NEM in the presence of either 150 mm NaCl plus 20 μm dl-TBOA or 150 mm KCl. After washing, d-[³H]aspartate uptake was measured. Results represent the mean ± S.E. (error bars) of at least three experiments performed in triplicates and are given as a percentage of the uptake activity of samples preincubated in the same medium without NEM. A, the means of the two different preincubation media for each mutant were compared using Student's two-tailed t test. *, p < 0.05. B, the reactivity of the single cysteine at position 120 in the presence of either 150 mm NaCl plus 20 μm dl-TBOA or 150 mm KCl was determined at the indicated NEM concentrations.

FIGURE 6.

Effect of the composition of the external medium on the inhibition of A120C by NEM. Cells expressing the A120C mutant were preincubated for 5 min with or without 1 mm NEM in 150 mm choline chloride (ChCl)-, NaCl-, or KCl-containing solution. 20 μm dl-TBOA and 1 mm l-glutamate (Glu) were added as indicated. After washing, d-[³H]aspartate uptake was measured. Results represent the mean ± S.E. (error bars) of at least three experiments performed in triplicates and are given as a percentage of the transport activity of samples preincubated in the same medium without NEM (control). The means of the different preincubation media were compared with that of the choline chloride-containing solution using a one-way analysis of variance with Dunnett's post hoc multiple comparison test. *, p < 0.01.

FIGURE 7.

Effect of the composition of the external medium on the inhibition of Y124C by MTSEA. The Y124C mutant was transiently expressed in HeLa cells. Subsequently, cells were preincubated for 5 min with or without 1 mm MTSEA in 150 mm choline chloride (ChCl)-, NaCl-, LiCl-, or KCl-containing solution. 20 μm dl-TBOA and 1 mm l-glutamate (Glu) were added as indicated. After washing, d-[³H]aspartate uptake activity was measured. Results represent the mean ± S.E. (error bars) of at least three experiments performed in triplicates and are given as a percentage of the transport activity of cells preincubated in the same medium without MTSEA. The means of the percentage activity for the different preincubation media were compared with that of choline chloride using a one-way analysis of variance with Dunnett's post hoc multiple comparison test. *, p < 0.05.

FIGURE 8.

Effect of MTSEA on single cysteine mutants. The indicated mutants were expressed in HeLa cells, followed by preincubation for 5 min in the presence or absence of 2.5 mm MTSEA in medium containing either 150 mm NaCl and 20 μm dl-TBOA or 150 mm KCl. After washing, d-[³H]aspartate transport was measured. Results represent the mean ± S.E. (error bars) of at least three experiments performed in triplicates and are given as a percentage of the transport activity of cells preincubated in the same medium without MTSEA. The means of the percentage remaining activity in different preincubation media for each mutant were compared using Student's two-tailed t test.

FIGURE 9.

Effect of substrate and blocker on inhibition of Y124C in 3 mm sodium. A, the mutant was transiently expressed in HeLa cells, followed by preincubation for 5 min in the presence or absence of 1 mm MTSEA in medium containing the indicated sodium concentrations (iso-osmotic replacement of NaCl with choline chloride (ChCl)). B, preincubation medium containing 3 mm sodium and 147 mm choline or choline alone was supplemented with the following: 20 μm dl-TBOA, 1 mm l-glutamate (Glu), or 1 mm GABA as indicated. After washing, d-[³H]aspartate transport was measured. Results represent the mean ± S.E. (error bars) of at least three experiments performed in triplicates and are given as a percentage of the transport activity of cells preincubated in the same medium without MTSEA. Statistics were done using a one-way analysis of variance with Dunnett's post hoc multiple comparison test. *, p < 0.001.

FIGURE 10.

Effect of MTSEA and NEM on Y124C. The mutant was transiently expressed in HeLa cells, followed by preincubation for 5 min, in the presence or absence of 1 mm MTSEA or 2 mm NEM in choline chloride (ChCl)-, sodium chloride (NaCl)-, or potassium chloride (KCl)-containing solution. After washing, d-[³H]aspartate transport was measured. Results represent the mean ± S.E. (error bars) of at least three experiments performed in triplicates and are given as a percentage of the uptake activity of samples preincubated in the same medium without MTSEA or NEM. The means obtained under the different conditions were compared with that in the presence of choline, using a one-way analysis of variance with a post hoc Dunnett's multiple comparison test. *, p < 0.01.