An acidic amino acid transmembrane helix 10 residue conserved in the neurotransmitter:sodium:symporters is essential for the formation of the extracellular gate of the γ-aminobutyric acid (GABA) transporter GAT-1

Abstract

GAT-1 mediates transport of GABA together with sodium and chloride in an electrogenic process enabling efficient GABAergic transmission. Biochemical and modeling studies based on the structure of the bacterial homologue LeuT are consistent with a mechanism whereby the binding pocket is alternately accessible to either side of the membrane and which predicts that the extracellular part of transmembrane domain 10 (TM10) exhibits aqueous accessibility in the outward-facing conformation only. In this study we have engineered cysteine residues in the extracellular half of TM10 of GAT-1 and probed their state-dependent accessibility to sulfhydryl reagents. In three out of four of the accessible cysteine mutants, the inhibition of transport by a membrane impermeant sulfhydryl reagent was diminished under conditions expected to increase the proportion of inward-facing transporters, such as the presence of GABA together with the cotransported ions. A conserved TM10 aspartate residue, whose LeuT counterpart participates in a "thin" extracellular gate, was found to be essential for transport and only the D451E mutant exhibited residual transport activity. D451E exhibited robust sodium-dependent transient currents with a voltage-dependence indicative of an increased apparent affinity for sodium. Moreover the accessibility of an endogenous cysteine to a membrane impermeant sulfhydryl reagent was enhanced by the D451E mutation, suggesting that sodium binding promotes an outward-facing conformation of the transporter. Our results support the idea that TM10 of GAT-1 lines an accessibility pathway from the extracellular space into the binding pocket and plays a role in the opening and closing of the extracellular transporter gate.

FIGURE 1.

Translocation cycle of GAT-1 and LeuT structure. A, the outward facing empty transporter (out T⁻) binds sodium ions (step 1), followed by GABA (G) and chloride (step 2) to yield the loaded outward facing transporter. This is followed by occlusion of GABA and the cotransported ions (step 3), and subsequently the loaded transporter becomes inward facing (step 4). After release of GABA and the cotransported ions to the cytoplasm (step 5), the empty inward facing transporter (in T⁻) transits via the empty transporter, with its binding pocket occluded, to yield again the outward facing empty transporter (steps 6 and 7), and a new translocation cycle can begin. One of the possible scenarios for the observed transient currents could be that the outward-facing sodium-bound transporter (outT⁺_2Na) becomes occluded (step 8). As described under “Discussion,” the alternative scenario consists of steps 6 and 7, followed by the stabilization of (outT⁻) by sodium (step 1). The outward facing transporter can also bind sodium ions and the blocker (step 9), which “locks” it in the outward conformation and prevents additional conformational changes. The sodium concentration dependence of the capacitative transient currents has been reported to be cooperative (11) and therefore we assume that both sodium ions bind to the empty outward facing transporter before GABA and chloride. The order of binding and debinding (steps 2 and 5) is not indicated. B, LeuT structure (Protein Data Bank 2A65) showing three residues involved in the formation of the “thin” extracellular gate. Bundle helices TMs 1, 2, 6, 7, as well as TMs 3, 8, and 10 are shown as indicated. Arg-30 (TM1), Gln-250 (TM6), and Asp-404 (TM10) correspond to Arg-69, Gln-291, and Asp-451 of GAT-1, respectively. The bound leucine substrate (yellow) and the 2 sodium ions (spheres) are also shown. The figure was prepared using PyMOL software.

FIGURE 2.

Transport activity of cysteine mutants. GAT-1-C74A (control) and cysteine mutants at positions 445–459 in the C74A background were transiently expressed in HeLa cells, and sodium-dependent [³H]GABA transport was measured at room temperature for 10 min, as described under “Experimental Procedures.” The data are given in mean ± S.E. (error bars) of at least three separate experiments performed in quadruplicate.

FIGURE 3.

Effect of MTSET on transport activity of cysteine mutants. HeLa cells transiently expressing each of the indicated cysteine-mutants were preincubated for 5 min with transport solution containing 150 mm NaCl, with or without 1 mm MTSET, as described under “Experimental Procedures” followed by washing and [³H]GABA 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 quadruplicate. The means of the mutants were compared with those of C74A using a one-way analysis of variance with a post-hoc Dunnett's multiple comparison test (*, p < 0.05).

FIGURE 4.

Effect of MTSET on alanine and cysteine mutants. The indicated cysteine (open bars) and alanine (gray hatched bars) mutants were transiently expressed in HeLa cells, and the effect of preincubation with 1 mm MTSET on transport activity was determined as described in the legend of Fig. 3. Error bars indicate mean ± S.E.

FIGURE 5.

Effect of the composition of the external medium on the inhibition of C74A/K448C, C74A/Y452C, C74A/Y453C, and C74A/S456C by MTSET. The four indicated cysteine mutants were transiently expressed in HeLa cells. Cells were preincubated 5 min with or without MTSET in a 150 mm sodium chloride (NaCl)- or choline chloride (ChCl)-containing solution. SKF-89976A (30 μm) (SKF) and GABA (1 mm) were added as indicated. After washing, [³H]GABA uptake activity was measured. Results represent the mean ± S.E. (error bars) of at least three experiments performed at least in quadruplicates and are given as a percentage of the uptake activity in samples preincubated in the same medium but without MTSET. For every mutant, the means of ChCl, NaCl + GABA, and NaCl + SKF were compared with those of NaCl alone, using a one-way analysis of variance with a post-hoc Dunnett's multiple comparison test (*, p < 0.05). The concentrations of MTSET used were 1, 0.04, 0.03, and 1 mm for K448C, Y452C, Y453C, and S456C, respectively.

FIGURE 6.

Transport activity of Asp-451 and Tyr-452 mutants. GAT-1-WT (control), Asp-451, and Tyr-452 mutants as well as the indicated single mutants were transiently expressed in HeLa cells, and sodium-dependent [³H]GABA transport was measured at room temperature for 10 min, as described under “Experimental Procedures.” The data are given in mean ± S.E. (error bars) of at least three separate experiments performed in quadruplicate.

FIGURE 7.

Cell surface biotinylation of GAT-1-WT and Asp-451 mutants. HeLa cells expressing GAT-1-WT (WT) and the indicated mutants, as well as HeLa cells transfected with the vector alone (SK), were biotinylated and processed as explained in “Experimental Procedures.” The markers shown were run in the lane to the left of SK and contain “Prestained Protein Marker, Broad Range,” Cat. # P7708S from New England Biolabs.

FIGURE 8.

Sodium-dependent transient currents and GABA-induced steady-state currents by GAT-WT and D451E transporters. The membrane voltage of oocytes expressing GAT- 1-WT or D451E was stepped from a holding potential of −25 mV to voltages between −140 to +60 mV in 25-mV increments. Each potential was held clamped for 500 ms, followed by 500 ms of a potential clamped at −25 mV. All traces shown are from the same oocytes, which represent at least three repeats. A, transient currents, currents in 10 μm tiagabine and the indicated sodium concentration were subtracted from those in the same medium, in the absence of tiagabine. B, GABA-induced currents, currents in ND96 were subtracted from those in the same medium supplemented with 1 mm (GAT-1-WT) or 10 mm (D451E) of GABA. The dashed lines indicate zero current. Where not indicated, the external sodium concentration was 100 mm. C, fit of the charge movements to a Boltzmann distribution as a function of potential. The charge movements of oocytes expressing WT-GAT-1 in 100 mm sodium (■) and D451E in 30 mm sodium (●) are plotted as a function of the voltage. Charge movements were normalized to Q_max and were fit, using the Boltzman distribution non-linear-curve-fit function in Origin 6.1 (OriginLab Corporation). The Q_max values of WT and D451E were 28.2 ± 2.4 and 12.2 ± 1.0 nC, respectively. Data points are averaged from 3 oocytes for each transporter (where the S.E. is not visible, the error is smaller than the size of the symbols).

FIGURE 9.

Effect of MTSET on the transient currents of GAT-1-WT, D451E, and C74A/D451E. Transient currents of oocytes expressing GAT-1 WT, D451E, or C74A/D451E were measured using the voltage-jump protocol described in Fig. 8. Currents in 100 mm NaCl and 10 μm tiagabine were subtracted from those recorded in the absence of tiagabine before (Control) and after a 2 min treatment with 5 mm MTSET, as described in “Experimental Procedures.” Traces shown are from the same oocytes, which represent at least three repeats. The dashed lines indicate zero-current.

FIGURE 10.

Effects of MTSET on the transient currents of GAT-1-WT, D451E, and C74A/D451E and reversal by DTT. A–C, transient currents of oocytes expressing GAT-1 D451E were measured using the voltage-jump protocol described in Fig. 8. All traces shown are from the same D451E-expressing oocyte, which represents three repeats. Currents in 100 mm NaCl and 10 μm tiagabine were subtracted from those recorded in the absence of tiagabine, before (Control) and after treatment with 5 mm MTSET and also after a subsequent exposure of the MTSET-treated oocytes to 10 mm DTT as described in “Experimental Procedures.” The dashed lines indicate zero-current. D, charge movements following treatment with 5 mm MTSET were normalized to those before the exposure to the sulfhydryl reagent for GAT-1 WT, D451E, and C74A/D451E. The data are given as mean ± S.E. (error bars) of three oocytes.

FIGURE 11.

Effects of sodium on the lithium leak currents of GAT-1-WT and D451E. Lithium-dependent currents, obtained by subtracting the response in sodium at −140 mV, were normalized to the values for the wild type or mutant and recorded at the indicated sodium concentrations. The lithium concentration was 86.4 mm, and choline was used to maintain iso-osmolarity. The data represent mean ± S.E. from three oocytes for both WT (■) and D451E (●).

FIGURE 12.

Sodium dependence of GAT-1-WT and D451E. HeLa cells expressing GAT-1-WT (■) and D451E (●) were assayed for [³H]GABA transport as described under “Experimental Procedures.” Transport was carried out for 3 min for WT and 60 min for D451E at the indicated sodium concentrations with choline as the substituting ion. The data shown at the indicated sodium concentrations are normalized to those of GAT-1-WT at 150 mm sodium (no choline substitution) or D451E, respectively and are the means ± S.E. of at least three separate experiments performed in quadruplicate.