Substrate-induced unlocking of the inner gate determines the catalytic efficiency of a neurotransmitter:sodium symporter

Abstract

Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na(+)-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na(+)-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.

Keywords: amino acid transport; conformational change; dopamine transporter; fluorescence quenching; fluorescence spectroscopy; gating; mechanisms of membrane transport; membrane protein; monoamine transporter; neurotransmitter transport.

FIGURE 1.

Probing small scale conformational changes at the cytosolic face of LeuT. A, two-dimensional diagram of LeuT embedded in the membrane. The cysteine (E192C) introduced for labeling with the sulfhydryl-reactive fluorophore TMR maleimide is indicated in orange (LeuT E192C^TMR). Mutations introduced in the primary and secondary substrate-binding sites are show in green (Y108F, F253A, and F253L) and blue (L400S), respectively. The I359Q mutation, shown in red, introduced in the primary substrate site converts LeuT to a tryptophan transporter. The conserved residues involved in the intracellular interaction network (Arg⁵, Trp⁸, Tyr²⁶⁸, and Asp³⁶⁹) are outlined in teal. B, structure of LeuT (outward occluded) showing the position of Glu¹⁹² with TM1i and TM5i highlighted in green. Glu¹⁹² is positioned in close proximity to the conserved intracellular interaction network. C, chemical structure of TMR maleimide that was conjugated to E192C at the cytosolic end of TM5.

FIGURE 2.

Purified and fluorescently labeled LeuT E192C (LeuT E192C^TMR) retains WT-like substrate binding activity. A, representative SDS-PAGE showing the purity of LeuT E192C^TMR eluted from nickel immobilized-metal affinity chromatography. Left, fluorescence scan showing specific TMR labeling of eluted LeuT E192C (“E”) and that unconjugated TMR is removed in the wash step (“W”). Right, the same gel stained with Coomassie Blue. B, saturation binding of [³H]leucine in 200 mm NaCl assessed in the SPA for WT LeuT (circles) and LeuT E192C^TMR (squares). Data are fitted to a single site model, and equilibrium binding constants are shown in Table 1. Data points are means ± S.E. (error bars) from four independent experiments performed in duplicates. C, specific accumulation of 1 μm [³H]alanine in liposomes reconstituted with WT LeuT (circles) or LeuT E192C^TMR (squares). Data points are duplicate determinations from a representative experiment. D, competition SPA binding of 10 nm [³H]leucine to LeuT E192C^TMR with leucine in the absence (squares) or presence of 200 mm KI (triangles). Data points are means ± S.E. (error bars) from two independent experiments performed in at least duplicates.

FIGURE 3.

Leucine induces a Na⁺-dependent increase in the aqueous accessibility of TMR bound to E192C (LeuT E192C^TMR). A and B, TMR maleimide fluorescence emission spectra in the presence of increasing concentrations of the hydrophilic quencher KI, normalized to maximum fluorescence intensity in the absence of KI (dashed line). A, fluorescence from unconjugated dye was quenched concentration-dependently by iodide. B, conjugation of TMR to E192C in LeuT (LeuT E192C^TMR) substantially decreased iodide quenching of TMR fluorescence. C, Stern-Volmer plots of quenching data in A and B showing that F₀/F (fluorescence normalized to intensity in the absence of KI) is linearly dependent on the KI concentration but that the slope (the Stern-Volmer quenching constant, K_SV) is substantially lower for TMR conjugated to E192C (LeuT E192C^TMR) (black circles) compared with unconjugated TMR (purple circles). D, iodide quenching of LeuT E192C^TMR in 200 mm NaCl in the absence of substrate (black circles) or presence of increasing fixed concentrations of leucine (blue squares, 100 nm; blue triangles, 1 μm; blue inverted triangles, 10 μm; blue diamonds, 100 μm; blue circles, 1 mm). E, iodide quenching of LeuT E192C^TMR in 200 mm KCl in the absence of substrate (black circles) or presence of increasing fixed concentrations of leucine (blue diamonds, 100 μm; blue circles, 1 mm). F, K_SV values obtained from D and E plotted as a function of leucine concentration, showing that leucine in 200 mm Na⁺ (filled squares) induces a concentration-dependent and saturable increase in iodide quenching of LeuT E192C^TMR. The response is abolished in the absence of Na⁺ (200 mm KCl; open squares). Data points are means ± S.E. (error bars) from four to five independent experiments.

FIGURE 4.

Kinetics of the LeuT E192C^TMR fluorescence quenching response and effects of CMI. A, fluorescence time course experiment monitoring the rate of change in LeuT E192C^TMR fluorescence upon addition of 100 μm leucine (arrow) in the presence of 200 mm KI. Data points are individual fluorescence recordings from two independent experiments normalized to the average steady-state fluorescence prior to leucine addition with control conditions (200 mm KCl) subtracted. Data are fitted to a two-phase exponential decay function. A.U., arbitrary units. B, addition of 100 μm CMI does not induce a significant increase in iodide quenching when the primary substrate site has no substrate bound. However, 100 μm CMI significantly potentiates the quenching response to leucine (1 mm) by decreasing the substrate off-rate. Note that fluorescence buffer containing Tris/MES (pH 7.50) instead of Tris-HCl was used to improve the solubility of CMI. Data points are percent change in the Stern-Volmer constant, K_SV (means ± S.E. (error bars) from three to five individual measurements; ***, p < 0.001; n.s., not significant; unpaired t test).

FIGURE 5.

Specificity and directionality of the LeuT E192C^TMR quenching response. A and B, KI quenching experiments on LeuT I191C^TMR or LeuT E192C^TMR. A and B, Stern-Volmer constants (K_SV) obtained from KI quenching experiments with the purified and TMR-labeled single cysteine LeuT mutants. K_SV values determined in a low sodium buffer (1 mm NaCl and 199 mm KCl), in a high sodium buffer (190 mm NaCl and 10 mm KCl), or in the presence of 1 mm leucine (in 190 mm NaCl and 10 mm KCl) are shown. Data points are means ± S.E. (error bars) from four to six independent experiments. Neither Na⁺ nor leucine causes a significant change in quenching of LeuT I191C^TMR. In a parallel series of experiments with LeuT E192C^TMR, leucine but not Na⁺ causes a marked increase in iodide quenching (***, p < 0.001). C, D, and E, leucine binding to LeuT E192C^TMR induces a conformation where TMR is less exposed to hydrophobic quencher molecules, incorporated in the detergent micelle. C, diagram showing the hydrophilic quencher iodide (I⁻; yellow circles) and the chemical structure of the hydrophobic quencher molecule TEMPO (red hexagons). TEMPO localizes to the hydrophobic environment of the detergent micelle and is expected to produce a higher degree of fluorescence quenching if TMR becomes more buried. In contrast, I⁻ exerts a higher degree of quenching when TMR exposure to the aqueous environment is increased. D, Stern-Volmer plots obtained from quenching of LeuT E192C^TMR fluorescence by the hydrophobic quencher TEMPO. Stern-Volmer relationships were determined in a low sodium buffer (inverted triangles; 1 mm NaCl and 199 mm KCl), in a high sodium buffer (circles; 190 mm NaCl and 10 mm KCl), or in the presence of 1 mm leucine (squares; in 190 mm NaCl and 10 mm KCl). E, Stern-Volmer constants (K_SV) obtained from the experiments shown in D. Data points are means ± S.E. (error bars) from four individual experiments (*, p < 0.05; unpaired t test).

FIGURE 6.

The coordinated rearrangements of TM1i and TM5i in the conformational transition predicted from the simulations. a, comparison in the WT LeuT of the inward-closed state (with TM1i and TM5i colored in green) and the modeled state (with TM1i and TM5i colored in cyan) resulting from the tMD/uMD study (see text). The conserved salt bridge between Arg⁵ and Asp³⁶⁹ is disrupted during the transition from the inward-closed to the modeled state. b, quantification of the modeled TM5i movement. The graph shows the probability distribution of the angle between TM1i and TM5i for WT LeuT (dashed line) and LeuT E192C^TMR (solid line) in the inward-closed (green) and modeled state (cyan). The probability of LeuT assuming a conformation with a larger angle between TM1 and TM5 is markedly increased in the modeled state for both WT LeuT and LeuT E192C^TMR. c and d, predicted conformation of TMR conjugated to E192C in the inward-closed state (c) and in the modeled state (d). The color scheme in c and d is the same as in a and b. In the modeled state, rotation of TM5i results in a movement of TMR attached to E192C (colored in magenta) from the lipid phase to the aqueous environment. e, quantification of TMR solvent exposure in the LeuT E192C^TMR conformations. Probability distribution of the solvent-accessible surface area (SASA) for TMR attached to E192C, showing a clear increase in the probability of the fluorophore to have a higher degree of solvent exposure in the modeled state compared with the inward-closed state.

FIGURE 7.

Conformational coupling to the inner gate depends on substrate binding to S1. A, Stern-Volmer plots of KI quenching data for the S1 substrate-binding site mutant LeuT E192C^TMR/Y108F. Experiments were conducted in 200 mm NaCl in the absence of substrate (black circles) or presence of increasing fixed concentrations of leucine (blue squares, 100 nm; blue triangles, 1 μm; blue inverted triangles, 10 μm; blue diamonds, 100 μm; blue circles, 1 mm). B, K_SV values obtained from A plotted as a function of leucine concentration. The dashed line shows data for LeuT E192C^TMR taken from Fig. 2F. C, Stern-Volmer plots of KI quenching data for the S2 substrate-binding site mutant LeuT E192C^TMR/L400S. Experiments were performed as described in A. D, K_SV values obtained from C plotted as a function of leucine concentration. The dashed line shows data for LeuT E192C^TMR taken from Fig. 2F. E and F, Stern-Volmer plots of KI quenching data for the S1 substrate-binding site mutants LeuT E192C^TMR/F253A and LeuT E192C^TMR/F253L. Experiments were performed as described in A. G and H, K_SV values obtained from E and G plotted as a function of leucine concentration. The dashed line shows data for LeuT E192C^TMR taken from Fig. 2F. By revealing a right shift in dose-response curves (EC₅₀ values given in Table 1) for the S1 mutants Y108F, F253A, and F253L but not for L400S, the data support the importance of leucine binding to S1 for the conformational response. Data points are means ± S.E. (error bars) of three to four independent experiments using protein from two separate preparations. For F253L, data points are means ± S.E. (error bars) of two to three independent experiments.

FIGURE 8.

The substrate-induced increase in iodide quenching of LeuT E192C^TMR is inversely correlated to the maximum attainable uptake rate of the substrate. A, chemical structures of the neutral amino acids used for the experiments in B–G. B, K_SV values obtained from KI quenching of LeuT E192C^TMR in 200 mm NaCl plotted against the indicated concentrations of tryptophan. No significant quenching response was observed. C, K_SV values obtained from KI quenching of LeuT E192C^TMR/I359Q in 200 mm NaCl plotted against the indicated concentrations of tryptophan, showing that introduction of I359Q enabled a Na⁺-dependent tryptophan-induced quenching response. D, K_SV values obtained from KI quenching of LeuT E192C^TMR in 200 mm NaCl plotted against the indicated concentrations of alanine. Alanine induced a concentration-dependent increase in quenching, but the magnitude of the response was smaller than that of leucine. E, maximum KI quenching response for the indicated amino acids. The experiment was performed using a 1 mm concentration of the amino acids in 200 mm NaCl (filled bars) or 200 mm KCl (empty bars). Data points are means ± S.E. (error bars) from three to five independent experiments. Note that fluorescence buffer containing Tris/MES (pH 7.50) instead of Tris-HCl was used to improve the solubility of the tested amino acids and that similar results were obtained in Tris-HCl buffer except with a smaller range of the individual K_SV values. F, uptake experiments on reconstituted LeuT using [³H]alanine (red symbols), [³H]valine (purple), [³H]isoleucine (green), [³H]methionine (yellow), and [³H]leucine (blue). Data points are specific uptake in nmol/min/mg of protein given as means ± S.E. (error bars) from three independent experiments. Kinetic constants are shown in Table 4. Aa, amino acid. G, the catalytic efficiencies (k_cat/K_m) for [³H]alanine, [³H]valine, [³H]isoleucine, [³H]methionine, and [³H]leucine are inversely correlated with the maximal quenching responses shown in E.