A conserved salt bridge between transmembrane segments 1 and 10 constitutes an extracellular gate in the dopamine transporter

Abstract

Neurotransmitter transporters play an important role in termination of synaptic transmission by mediating reuptake of neurotransmitter, but the molecular processes behind translocation are still unclear. The crystal structures of the bacterial homologue, LeuT, provided valuable insight into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the "thin gate" formed by an interaction between Arg-30 and Asp-404. In the human dopamine transporter (DAT), the corresponding residues are Arg-85 and Asp-476. Here, we present results supporting the existence of a similar interaction in DAT. The DAT R85D mutant has a complete loss of function, but the additional insertion of an arginine in opposite position (R85D/D476R), causing a charge reversal, results in a rescue of binding sites for the cocaine analogue [(3)H]CFT. Also, the coordination of Zn(2+) between introduced histidines (R85H/D476H) caused a ∼ 2.5-fold increase in [(3)H]CFT binding (Bmax). Importantly, Zn(2+) also inhibited [(3)H]dopamine transport in R85H/D476H, suggesting that a dynamic interaction is required for the transport process. Furthermore, cysteine-reactive chemistry shows that mutation of the gating residues causes a higher proportion of transporters to reside in the outward facing conformation. Finally, we show that charge reversal of the corresponding residues (R104E/E493R) in the serotonin transporter also rescues [(3)H](S)-citalopram binding, suggesting a conserved feature. Taken together, these data suggest that the extracellular thin gate is present in monoamine transporters and that a dynamic interaction is required for substrate transport.

Keywords: Molecular Pharmacology; Monoamine Transporter; Neuroscience; Neurotransmitter Transport; Protein Structure; Structure-Function Relationship; Zinc Site Engineering.

FIGURE 1.

Presumed localization of the investigated residues in DAT. A, two-dimensional schematic representation of the human DAT. Red circles indicate the locations of the two investigated residues, Arg-85 and Asp-476, in TM1 and TM10, respectively. The open circles represent amino acid residues ordered in TMs and loop regions. The presumed glycosylation sites are indicated with small filled circles. B, molecular model of the human DAT based on the LeuT outward facing closed structure with dopamine (spheres in blue (carbons), gray (hydrogens), and red (oxygens)) docked in the center of the protein. Only a fraction of the protein is shown (gray helices and loops). The two presumed gating residues, Arg-85 and Asp-476, are depicted as orange sticks. The residues presumably closing the dopamine accessibility from the extracellular environment, Asp-79 and Tyr-165, are shown as thin black sticks. TM11 and 12 are removed for clarity.

FIGURE 2.

Reversal of the presumed salt bridge residues can partly rescue DAT function. A, [³H]DA uptake capacity of the DAT WT and mutants with either a single (R85D and D476R) or both charges (R85D/D476R) reversed. Neither of the mutants were able to accumulate [³H]DA distinguishable from the background signal. B, binding of the high affinity cocaine analogue [³H]CFT to DAT WT and mutants. For the DAT R85D mutant, the binding of [³H]CFT was indistinguishable from the background signal. Both the DAT D476R and DAT R85D/D476R gave B_max values ∼10% of the WT signal (Table 1). C, transporter surface expression as quantified by ELISA on intact COS7 cells transiently transfected with a DAT background construct that has a HA tag inserted into the second extracellular loop. The data are means ± S.E. with the DAT WT expression set as 100% in all experiments. The surface expression of the indicated DAT mutants were 22 ± 3, 21 ± 3, and 28 ± 5% for R85D, D476R, and R85D/D476R, respectively, relative to the WT transporter. D, inhibition of [³H]CFT binding by DA in DAT WT (black squares) and DAT R85D/D476R (red circles). DA is able to displace [³H]CFT binding both in the DAT WT (K_i = 0.91 (0.66; 1.26) μm, mean (S.E. interval), n = 5) and in the DAT R85D/D476R (K_i = 15 (11, 20) μm, mean (S.E. interval), n = 4). All experiments are performed in triplicate with a concentration range of CFT (0.1 nm to 100 μm) or DA (1 nm to 1 mm) in 12 determinations on intact COS7 cells transiently transfected with DAT WT or indicated mutant.

FIGURE 3.

Mutations to the proposed salt bridge do not permit Li⁺-sensitive leak in the transporter. A–D, I/V plots of steady-state cocaine sensitive leak currents in WT, R85D, D476R, and the double mutant R85D/D476R assessed in 130 mm NaCl (open black squares) and 130 mm LiCl (open red circles) means ± S.E. (n = 4–5). The inwardly rectifying leak current observed in WT when substituting NaCl with LiCl is not observed when mutating residues in the proposed outer gate (B–D). The I/V plots were generated in 20-mV steps from −100 to +40 mV.

FIGURE 4.

Functional analysis of the TM1/TM10 interaction by Zn²⁺ binding to the histidine substituted mutant DAT R85H-D476H. A, the molecular docking model of DAT from Fig. 1 here with inserted histidines in positions 85 and 476 (orange sticks). The distance between possible coordinating histidines, here exemplified as an interaction between the π (δ1) and τ (ϵ2) nitrogens, is 3.8 Å, which is within Zn²⁺ coordinating distance. B, the addition of Zn²⁺ to R85H/D476H (red circles) dose-dependently increases [³H]CFT binding to 243 ± 19% (mean ± S.E., n = 8) in 1 mm Zn²⁺ relative to no Zn²⁺ present. The effect was observed when applying subsaturating concentrations of [³H]CFT together with increasing concentrations of Zn²⁺ up to a final concentration of 1 mm. No effect of Zn²⁺ on [³H]CFT binding was observed on either R85H (black squares) or D476H (black triangles) single mutants (n = 7). C, Zn²⁺ decreases off rate of bound [³H]CFT to the R85H/D476H mutant. The dissociation rate constant (K) for [³H]CFT from R85H/D476H shows a 5-fold decrease by the addition of 200 μm Zn²⁺, from 0.076 ± 0.0038 min⁻¹ (black circles) to 0.015 ± 0.0015 min⁻¹ (cyan squares) (mean ± S.E., n = 7). No effect of Zn²⁺ on dissociation was observed in the R85H, D476H, or H193K background mutants (data in text). D, transport of [³H]DA by R85H/D476H is inhibited by the addition of Zn²⁺. The uptake capacity by R85H/D476H is markedly decreased relative to the DAT H193K background construct but still within the detection limit of the assay (Table 3). The addition of Zn²⁺ to R85H/D476H (red circles) results in a dose-dependent inhibition of [³H]DA uptake (K_i = 2.6 [2.1;3.1] μm, mean[S.E. interval], n = 3). The effect of Zn²⁺ on the single mutants R85H (squares) and D476H (triangles) were indistinguishable from the Zn²⁺ effect on the background mutant H193K reported previously (36). All mutations were performed in the background of the Zn²⁺-insensitive mutant DAT H193K. The data are means ± S.E. performed in triplicate on COS7 cells transiently transfected with the indicated mutants.

FIGURE 5.

Mutation of Arg-85 or Asp-476 increases accessibility to Cys-159. Insertion of a cysteine in position 159 into the MTSET-insensitive DAT background E2C (C90A/C306A), DAT E2C I159C, can be used as a tool to probe for accessibility for Cys-159 to the extracellular environment. Preincubation with MTSET (0.5 mm, 5 min) to DAT E2C I159C results in an inhibition of [³H]DA uptake of 12.0 ± 1.1%. The additional mutation of R85H, D476N, or D476H resulted in a significantly increased [³H]DA uptake inhibition by MTSET to 43 ± 8, 37 ± 10, and 32 ± 7%, respectively. The data are means ± S.E. (n = 5–8) performed in triplicate on COS7 cells transiently transfected with the indicated mutants.

FIGURE 6.

The TM1/TM10 interaction is also present in SERT. Mutation of the aligned position to Asp-476 in SERT (E493R) completely abolishes any detectable [³H]S-CIT binding. Mutation of R104E in SERT E493R (SERT R104E/E493R) restores binding to ∼30% of SERT WT levels ([³H]S-CIT B_max (fmol/100 μl of membrane) SERT WT = 468 ± 32; SERT R104E/E493R = 152 ± 10). Mutation of R104E alone reduces [³H]S-CIT binding to an approximately similar level as the double mutant (SERT R104E, B_max = 468 ± 32 fmol/100 μl of membrane). All data are shown as B_max values of three experiments performed in triplicate as described under “Experimental Procedures.”