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J. Biol. Chem., Vol. 279, Issue 5, 3228-3238, January 30, 2004

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Identification of Intracellular Residues in the Dopamine Transporter Critical for Regulation of Transporter Conformation and Cocaine Binding^*

Claus Juul Loland, Charlotta Grånäs, Jonathan A. Javitch, and Ulrik Gether¶

From the Molecular Neuropharmacology Group, Department of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen, Denmark and the Center for Molecular Recognition and the Departments of Psychiatry and Pharmacology, Columbia University College of Physicians & Surgeons, New York, New York 10032

Received for publication, May 7, 2003 , and in revised form, October 9, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recently we showed evidence that mutation of Tyr-335 to Ala (Y335A) in the human dopamine transporter (hDAT) alters the conformational equilibrium of the transport cycle. Here, by substituting, one at a time, 16 different bulky or charged intracellular residues, we identify three residues, Lys-264, Asp-345, and Asp-436, the mutation of which to alanine produces a phenotype similar to that of Y335A. Like Y335A, the mutants (K264A, D345A, and D436A) were characterized by low uptake capacity that was potentiated by Zn²⁺. Moreover, the mutants displayed lower affinity for cocaine and other inhibitors, suggesting a role for these residues in maintaining the structural integrity of the inhibitor binding crevice. The conformational state of K264A, Y335A, and D345A was investigated by assessing the accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of a cysteine engineered into position 159 (I159C) in transmembrane segment 3 of the MTSET-insensitive "E2C" background (C90A/C306A). Unlike its effect at the corresponding position in the homologous norepinephrine transporter (NET I155C), MTSET did not inhibit uptake mediated by E2C I159C. Furthermore, no inhibition was observed upon treatment with MTSET in the presence of dopamine, cocaine, or Zn²⁺. Without Zn²⁺, E2C I159C/K264A, E2C I159C/Y335A, and E2C I159C/D345A were also not inactivated by MTSET. In the presence of Zn²⁺ (10 µM), however, MTSET (0.5 mM) caused up to 60% inactivation. As in NET I155C, this inactivation was protected by dopamine and enhanced by cocaine. These data are consistent with a Zn²⁺-dependent partial reversal of a constitutively altered conformational equilibrium in the mutant transporters. They also suggest that the conformational equilibrium produced by the mutations resembles that of the NET more than that of the DAT. Moreover, the data provide evidence that the cocaine-bound state of both DAT mutants and of the NET is structurally distinct from the cocaine-bound state of the DAT.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The dopamine transporter (DAT)¹ is responsible for the rapid re-uptake of dopamine released upon neuronal stimulation (1–3). In this way, the transporter controls the availability of dopamine in the synaptic cleft and thereby plays a key role in regulating the broad spectrum of physiological function mediated by dopamine (1–3). The critical physiological role of DAT has been illustrated by targeted disruption of the DAT gene in mice, which resulted in multiple deficits and profoundly altered dopaminergic neurotransmission (4, 5). Furthermore, the DAT has been the focus of much attention because it represents the principle target for the action of widely abused psychostimulants such as cocaine and amphetamine (3, 6).

The DAT is a prototypic member of the class of Na⁺/Cl^–-dependent transporters, along with neurotransmitter transporters, including the norepinephrine (NET), serotonin (SERT), -aminobutyric acid, and glycine transporters (3, 6). This family of transporters is believed to contain 12 putative transmembrane segments (TMs) connected by alternating extracellular and intracellular loops (ICLs) with an intracellular location of the N and C termini (Fig. 1) (3, 6). A high-resolution structure is not yet available for DAT or any related transporter, and our insight into the packing of the 12 helices remains limited (3, 6). The first series of proximity relationships have only quite recently been described in the tertiary structure of the human DAT (hDAT) (7–9). Interestingly, an increasing amount of evidence suggest that the DAT exists in the membrane as an oligomeric structure, but the functional significance of this still needs to be clarified (10–13).

View larger version (59K): [in this window] [in a new window]   FIG. 1. Two-dimensional schematic representation of the human dopamine transporter. The residues highlighted on the intracellular side are residues mutated to alanines in the present study (enlarged gray circles with black letters or black circles with white letters). The previously described mutation Y335A is indicated by a white letter in an enlarged gray circle (20). Position Ile-159, which was mutated to cysteine and alanine is also highlighted (black letter in enlarged white circle). The residues on the extracellular side coordinating Zn2+ binding to the endogenous Zn2+ binding site in hDAT are shown as enlarged gray circles with white letters (7, 8).

A prerequisite for such an alternating access model is the existence of both external and internal "gates," that is protein domains that undergo significant conformational changes during the transport cycle and are capable of occluding access to the substrate binding site from the extracellular or intracellular environment, respectively. Currently we know rather little about these putative gating domains, although the application of the substituted cysteine accessibility method has identified several conformationally active regions of the transporter (15–19). We also know very little about the molecular mechanisms governing the equilibrium between the distinct conformational states in the transport cycle, although this must be tightly regulated for proper transporter function. Interestingly, we have recently identified a tyrosine (Tyr-335) in the third ICL of hDAT that may play a key role in regulating this conformational equilibrium (20). This inference was primarily based on the observation that mutation of the tyrosine alters completely the effect of Zn²⁺ at the previously identified endogenous Zn²⁺ binding site in the hDAT (Refs. 7 and 8 and Fig. 1). In the WT, Zn²⁺ acts as a potent non-competitive inhibitor of transport via interaction with three residues on the extracellular face of the transporter (Fig. 1) (7). In marked contrast, in the Tyr-335 mutant (Y335A) this inhibitory Zn²⁺ switch is converted into a stimulatory Zn²⁺ switch, i.e. the transporter only displays efficient uptake in the presence of Zn²⁺ (20). We inferred that mutation of Tyr-335 produced a constitutive shift in the distribution of conformational states in the transport cycle and that this shift could be reversed in part by Zn²⁺. A major alteration of the conformational equilibrium in Y335A was further supported by a substantial decrease of up to 150-fold in the apparent affinity for cocaine and related inhibitors and a parallel increase of up to 20-fold in the apparent affinity for substrates (20).

We proposed that Tyr-335 is part of a network of intramolecular interactions, possibly in the gating domains themselves, that is important for stabilizing the transporter in a conformational state that maintains the structural integrity of the inhibitor binding site and to which extracellular substrate can bind and initiate transport (20). If this hypothesis is correct, mutation of other residues might display a similar phenotype as the Y335A mutation, due to their participation in the same network of regulatory intramolecular interactions. In this study, we have sought to test this hypothesis and accordingly to identify additional intracellular residues with a similar phenotype as Y335A. In agreement with our prediction, we identify three residues of 16 mutated charged or bulky residues on the predicted intracellular face of the transporter with such a phenotype. These residues are either in the same loop as Tyr-335, ICL 3, or in the "adjacent" ICL 2 and ICL 4. Furthermore, we establish a structural read-out of the conformational state of the mutant transporters by using the reactivity of a cysteine engineered into position 159 in TM 3. This position was chosen based on recent observations by Rudnick and co-workers (17) in the homologous SERT and NET indicating that the accessibility of the corresponding residue (position 155 in NET and 179 in SERT) to the positively charged sulfhydryl-reactive compound MTSET ([2-(trimethylammonium)-ethyl]-methanethiosulfonate) is dependent on whether the transporter assumes an outward facing conformation or an inward facing conformation. Consistent with our hypothesis, the accessibility data provide direct structural support for an alteration in the conformational equilibria of the mutant transporters that can be reversed in part by Zn²⁺

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Site-directed Mutagenesis—We generated a synthetic hDAT (syn-DAT) gene that encodes a protein with an amino acid sequence identical to that of hDAT WT, but the nucleotide sequence was altered to increase the number of unique restriction sites and to optimize codon utilization. The nucleotide sequence of this construct and its generation will be described elsewhere.² We have used this synthetic construct in previous work, and its expression and function are identical to DAT encoded by the wild type DAT gene (10, 21). The E2C background was generated by mutation in the synDAT background of Cys-90 and Cys-306 to alanine. These two endogenous cysteines were previously demonstrated to be accessible to charged sulfhydryl reagents applied extracellularly (15). This construct was designated "E2C," because the two extracellular cysteines were mutated. The synDAT gene and synDAT E2C gene were subcloned into pcDNA3 (Invitrogen) using the restriction sites for KpnI and XbaI. All mutations were generated by two-step PCR mutagenesis using Pfu polymerase (Promega, Madison, WI) with either synDAT WT or synDAT E2C as templates. The mutant PCR fragments were digested with the appropriate enzymes and cloned into the expression vector, purified by agarose gel electrophoresis and ligated into the vector using the TaKaRa ligation kit (Takara Bio Inc., Shiga, Japan). All mutations were confirmed by restriction enzyme mapping and DNA sequencing using an ABI 310 automated sequencer according to the manufacturer's instructions.

Indexing of Residues—A generic numbering scheme for amino acid residues in the family of Na⁺/Cl^–-coupled transporters has recently been proposed to facilitate direct comparison of positions between the individual members of the transporter family (22). According to this scheme the most conserved residue in each transmembrane segment has been given the number 50, and each residue is numbered according to its position relative to this conserved residue. For example, 1.55 indicates a residue in TM1 five residues carboxyl-terminal to the most conserved residue in this TM (Trp1.50). For the DAT, the most conserved residues in each transmembrane domain is as follows: TM1, Trp-84; TM2, Pro-112; TM3, Tyr-156; TM4, Cys-243; TM5, Leu-287; TM6, Gln-317; TM7, Phe-356; TM8, Phe-412; TM9, Gly-468; TM10, Gly-500; TM11, Pro-529; TM12, Gly-561. The generic numbers for residues mutated in this study are (in superscript): Arg-60^1.26, Glu-61^1.27, Phe-123^2.61, Ile-159^3.53, Lys-260^4.67, Lys-264^5.27, Tyr-335^6.68, Tyr-343^7.28, Arg-344^6.29, Asp-345^6.30, Asp-421^8.59, Glu428^8.66, Asp-436^9.18, Glu-437^9.19, Phe-438^9.20, Asp-507^10.57, Tyr-519^11.40, and Tyr-578^12.67. In E2C we have mutated Cys-90^1.56 and Cys-306^6.39.

Expression in COS-7 Cells—COS-7 cells were maintained at 37 °C in 10% CO₂ in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 0.01 mg/ml gentamicin (all from Invitrogen). WT and mutant transporters were transiently transfected into COS-7 cells by the calcium phosphate precipitation method as previously described (23, 24).

[³H]Dopamine Uptake Experiments—Uptake assays were performed as modified from Giros et al. (25) using 2,5,6-[³H]dopamine (7–21 Ci/mmol) (Amersham Biosciences). The uptake assays were carried out 2 days after transfection of transiently transfected COS-7 cells. Twenty hours after transfection, the cells were plated in poly-D-lysine-coated 24- or 12-well dishes (1 or 3 x 10⁵ cells/well, respectively) depending on the expression level of the particular mutated transporter to achieve an uptake level of 5–10% of total added [³H]dopamine. Prior to the experiment, the cells were washed once in 500 µl of uptake buffer (25 mM HEPES adjusted to pH 7.4 upon addition of 130 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄,1mM L-ascorbic acid, 5 mM D-glucose, and 1 µM of the catechol-O-methyltransferase inhibitor Ro 41-0960 (Sigma) at room temperature. Non-labeled compounds (dopamine, norepinephrine, Zn²⁺, CFT (2-carbomethoxy-3-(4-fluorophenyl)tropane), cocaine, or GBR 12,909) were added to the cells at the indicated concentrations. When the cells were co-incubated with 10 µM Zn²⁺ and the indicated non-labeled compound, Zn²⁺ was added to the uptake media prior to the addition of non-labeled compound. Uptake was initiated by addition of 50–90 nM [³H]dopamine in a final volume of 500 µl of uptake buffer. After 10 min of incubation at 37 °C, the cells were washed twice with 500 µl of ice-cold uptake buffer, lysed in 300 µl of 1% SDS, and left for 1 h at 37 °C. All samples were subsequently transferred to 24-well counting plates (PerkinElmer Life Sciences) followed by addition of 600 µl of Opti-phase HiSafe 3 scintillation fluid (PerkinElmer Life Sciences). The samples were counted in a Wallac Tri-Lux scintillation counter (PerkinElmer Life Sciences). Nonspecific uptake was determined in the presence of 1 mM dopamine (RBI, Natick, MA). All determinations were performed in triplicate.

[³H]Norepinephrine Uptake Experiments—Assays were performed as described above for [³H]dopamine uptake experiments, except that the [³H]dopamine was substituted by 40–80 nM [7,8-³H]norepinephrine (6–12 Ci/mmol) (Amersham Biosciences).

Surface Biotinylation—Biotinylation of cell surface proteins was performed essentially as described (26) by reaction with the membrane impermeant amine-specific biotinylating reagent sulfo-NHS-SS-biotin (Pierce). Transfected COS-7 cells were seeded in poly-D-lysine-coated 100-mm cell culture dishes (Corning) at 2.5 x 10⁶ cells/dish and grown for 24 h before the experiment. The cells were washed with ice-cold phosphate-buffered saline/Ca-Mg (pH 7.3), before treatment with sulfo-NHS-SS-biotin (1.5 mg/ml) at 4 °C for 40 min in phosphate-buffered saline/Ca-Mg followed by three washes with TBS (0.05 M Tris, pH 7.4, 0.3 M NaCl) and three washes with phosphate-buffered saline/Ca-Mg. The cells were solubilized in 1 ml of solubilization buffer (25 mM Tris, pH 7.5, with 1.0% Triton X-100, 150 mM NaCl, 1 mM EDTA, 5 mM N-ethylmaleimide, 200 µM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture tablet (Roche Diagnostics)), scraped off and left for 30 min at 4 °C with constant shaking. Lysates were centrifuged at 20,000 x g for 30 min at 4 °C, and the protein concentration in the supernatants was determined using the BCA assay kit (Pierce). Monomeric avidin beads (175 µl) (Pierce) were added to 500 µg of total protein from each sample. The volume was adjusted to 1.0 ml with solubilization buffer and the samples were incubated for 1 h at room temperature. The beads were washed four times with 800 µl of solubilization buffer, before elution with 50 µl of 2x loading buffer (100 mM Tris-HCl, pH 6.8, 20% glycerol, 10% SDS, 0.1 M dithiothreitol, and 0.2% bromphenol blue) for 30 min at 37 °C. The eluates (25 µl) were resolved by SDS-PAGE (10%) and immunoblotted with the rat monoclonal antibody MAB 369 directed against the NH₂ terminus of the hDAT (Chemicon) diluted 1:1000. Immunoreactive bands were visualized using goat anti-rat horseradish peroxidase-conjugated secondary antibody (1:10,000) and Pico Luminescence (Pierce). Quantification of bands was performed by densitometry measures using Scion Image (Scion, Frederick, MD) using film exposures that were in the linear range.

MTSET Experiments—Two days after transfection, cells seeded in 12- or 24-well plates were washed once with 500 µl of uptake buffer (see above). The cells were subsequently incubated with 0.5 mM MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate) (Toronto Research Chemicals, Toronto, Canada) (unless another concentration is indicated) at 37 °C for 10 min. The stock MTSET solution was freshly prepared in H₂O and immediately diluted 10-fold by application to the transfected cells into a final volume of 500 µl of uptake buffer. After incubation, the cells were washed twice in 500 µl of uptake buffer before initiation of [³H]dopamine uptake, performed as described above. The effects of substrates and blockers on MTSET reactivity were investigated by the addition of 100 µM dopamine or 10 µM cocaine immediately prior to the addition of MTSET. Subsequently, the cells were washed twice in 500 µl of uptake buffer before initiation of [³H]dopamine uptake assay.

Data Calculations—Uptake data and binding data were analyzed by nonlinear regression analysis using Prism 3.0 from GraphPad Software, San Diego, CA. The IC₅₀ values used in the estimation of K_m for uptake were calculated from means of pIC₅₀ values and the S.E. interval from the pIC₅₀ ± S.E. The K_I values were calculated from the IC₅₀ values using the equation K_I = IC₅₀/(1 + (L/K_m)), L = concentration of [³H]dopamine. One-way analysis of variance followed by Newman-Keuls multiple comparison post-hoc test was used for statistical comparisons of the response to MTSET treatment. To achieve an exact measure for the low specific transporter-mediated [³H]dopamine uptake for the E2C I159C/Y335A mutant with no Zn²⁺ present, an uptake experiment on mock-transfected COS-7 cells was performed in parallel. The nonspecific uptake in these cells was subtracted from the total uptake in E2C I159C/Y335A expressing cells. The nonspecific uptake was less than 20% of total uptake in E2C I159C/Y335A.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mutation of Selected Intracellular Residues in the hDAT—To identify residues that upon mutation might display the same phenotype as our previously described Tyr-335 to alanine mutation (Y335A), we selected 16 residues distributed throughout the intracellular domain of the hDAT (Fig. 1). First, we selected a series of tyrosines and phenylalanines that were either conserved in all or in many of the mammalian Na⁺/Cl^–-coupled transporters (Fig. 2): Phe-123 in ICL 1; Tyr-343 in ICL 3; Phe-438 in ICL 4; Tyr-519 in ICL 5; and Tyr-578 in the COOH terminus (for the generic number of these residues according to the numbering scheme proposed by Goldberg et al. see indexing under "Materials and Methods"). Next we selected a series of highly conserved charged residues because we hypothesized that such residues might be involved in important intramolecular interactions critical for maintaining the proper conformational equilibrium of the transport cycle. The selected residues included: Arg-60 and Glu-61 in the NH₂ terminus; Lys-260 and Lys-264 in ICL 2; Arg-344 and Asp-345 in ICL 3; Asp-421, Glu-428, Asp-436, and Glu-437 in ICL 4; and Asp-507 in ICL5. As is apparent from the alignment in Fig. 2, our selection of residues was representative but was not a comprehensive selection of all the conserved intracellular residues in the DAT with a bulky or charged side chain.

View larger version (102K): [in this window] [in a new window]   FIG. 2. Aligned sequences of putative ICLs from 12 Na+/Cl–-coupled transporters. Twelve Na+/Cl–-coupled transporters were aligned. Residues that are 100% conserved among the aligned sequences are shown in black. Functionally conserved residues are shown in gray. The residues mutated one at a time into alanines are listed with their respective positions in hDAT. Numbers for mutations causing a similar phenotype as the Y335A mutation are shown in bold italic.

View larger version (28K): [in this window] [in a new window]   FIG. 3. The effect of Zn2+ on [3H]dopamine uptake in hDAT WT and mutant transporters. Zn2+ inhibition of [3H]dopamine uptake in COS-7 cells transiently expressing hDAT WT (dark squares), or K264A (open circles), D345A (dark triangles), and D436A (open diamonds) mutant transporters, respectively. A, a representative experiment (out of at least three) to illustrate the potentiating effect of Zn2+ on mutant transporters. Note that the experiment was performed with a subsaturating concentration of [3H]dopamine causing the uptake of the mutant transporters to be relatively higher with increasing Zn2+ compared with WT because of the lower Km of the mutants (see Table I). Data are mean ± S.E. in triplicate determinations from a representative experiment. B, normalized values in percent of [3H]dopamine uptake in the absence of Zn2+ expressed as mean ± S.E. of five to seven experiments performed in triplicate.

View larger version (45K): [in this window] [in a new window]   FIG. 4. Surface expression of K264A and D345A compared with DAT wt and Y335A. A, representative Western blot showing surface biotinylation of COS-7 cells transiently expressing hDAT WT, K264A, D345A, or Y335A. Surface-biotinylated protein was purified using monomeric avidin beads and analyzed using SDS-PAGE followed by immunoblotting with the rat monoclonal antibody MAB 369 directed against the NH2 terminus of the hDAT (Chemicon) as described under "Materials and Methods." B, immunoreactive bands were quantified by densitometry measures using Scion Image (Scion, Frederick, MD). Data are mean ± S.E. of four independent experiments. Note that surface expression of Y335A compared with WT hDAT has previously been reported (20).

View larger version (22K): [in this window] [in a new window]   FIG. 5. Decreased apparent affinity for blockers of transport upon mutation of Lys-264 (K264A) and Asp-345 (D345A). A, normalized data showing inhibition of [3H]dopamine uptake by: A, CFT; B, cocaine; and C, GBR 12909. Experiments were performed in COS-7 cells expressing hDAT WT (dark squares), K264A (open circles), or D345A (dark triangle). Values are in percent of control (no inhibitor present) for each data set expressed as mean ± S.E. of three to eight experiments performed in triplicate.

[³H]Norepinephrine Uptake Versus [³H]Dopamine Uptake in Mutant Transporters—[³H]Dopamine uptake was compared with [³H]norepinephrine uptake in the three mutants (K264A, Y335A, and D345A) as well as in the hDAT WT and hNET WT. In parallel to our observations for [³H]dopamine, Zn²⁺ strongly potentiated [³H]norepinephrine uptake in the three mutants by increasing the V_max values up to 20-fold (Table IV). The K_m values for both [³H]dopamine and [³H]norepinephrine uptake were 3–4-fold lower in hNET than in hDAT (Table IV). Similarly, the K_m values for both [³H]dopamine and [³H]norepinephrine uptake were lower in all three mutants than in hDAT both in the presence and absence of Zn²⁺ (2–10-fold) (Table IV). The hNET displayed an 50% higher V_max for [³H]norepinephrine than for [³H]dopamine, whereas the hDAT displayed a similar V_max for both [³H]norepinephrine and [³ Curiously, the V_max values for [³ H]dopamine. H]norepinephrine tended in the mutants, as in the hNET and in contrast to the hDAT, to be larger than for [³H]dopamine in the mutants both in the absence and presence Zn²⁺ (Table IV).

The I159C mutation was introduced into a background hDAT construct (E2C) in which two extracellularly accessible cysteines had been mutated (C90A/C306A). E2C displayed little sensitivity to MTSET and uptake properties identical to the hDAT WT (Table III). As shown in Fig. 6, [³H]dopamine uptake was essentially indistinguishable from control cells after 10 min treatment with 0.5 mM MTSET of COS-7 cells transiently expressing E2C. Also at higher concentrations (up to 5 mM) we observed no significant inhibition of uptake (data not shown). Similar results were obtained for E2C when the MTSET reaction was performed in the presence of either 100 µM dopamine or 10 µM cocaine (Fig. 6). Addition of 10 µM Zn²⁺ also did not result in appreciable changes in MTSET sensitivity, either in the absence or presence of dopamine or cocaine (Fig. 6). Taken together, these data suggest that E2C is an appropriate background for the I159C mutation.

View larger version (14K): [in this window] [in a new window]   FIG. 6. Effect of MTSET treatment of mutant transporters on [3H]dopamine uptake. COS-7 cells transiently expressing the indicated constructs were treated with vehicle or 0.5 mM MTSET in the presence or absence of Zn2+ (10 µM) for 10 min in the presence of A,H2O; B, dopamine (DA); or C, cocaine. Data are shown as percent inhibition of [3H]dopamine uptake in the absence of MTSET (vehicle) for each individual condition (mean ± S.E. of three to four experiments performed in triplicate). *, significantly different from values in the absence of Zn2+ (p < 0.001, Newman-Keuls multiple comparison post-hoc test); **, significantly different from values in the absence of dopamine (p < 0.001, Newman-Keuls multiple comparison post-hoc test); ***, significantly different from values in the absence of cocaine (p < 0.001, Newman-Keuls multiple comparison post-hoc test).

The apparent discrepancy between our observations in DAT and the previous observations in NET was not because of differences in the experimental set-up, such as, for example, the use of different cells (COS-7 cells versus HeLa cells), because we also mutated Ile-155 in NET to cysteine and observed the same MTSET sensitivity of this mutant as previously reported (data not shown). Thus, the data strongly suggest that Ile-159 (DAT)/Ile-155 (NET) is considerably more accessible in NET than in DAT, despite the high degree of homology between the two transporters (70% sequence identity).

Notwithstanding the small effect of MTSET on E2C I159C, we did insert K264A, Y335A, and D345A into both the E2C background and the E2C I159C background. All these constructs were functional, as assessed by measurement of [³H]dopamine uptake (Table III and data not shown). E2C I159C/K264A, E2C I159C/Y335A, and E2C I159C/D345A were analyzed in detail and showed uptake properties almost identical to those observed for the mutants in the WT background including strong potentiation by low micromolar concentrations of Zn²⁺ (Tables II and III). Treatment of the mutants lacking the cysteine in position 159 (E2C K264A, E2C Y335A, and E2C D345A) with MTSET (0.5 mM) had little effect on [³H]dopamine uptake both in the absence and presence of dopamine or cocaine (Fig. 6). Also in the presence of Zn²⁺, MTSET treatment had only insignificant effects on [³H]dopamine uptake in E2C K264A, E2C Y335A, and E2C D345A (Fig. 6). A different picture was observed, however, in E2C I159C/K264A, E2C I159C/Y335A, and E2C I159C/D345A. In the absence of Zn²⁺, MTSET still had little or no effect on [³H]dopamine uptake, but addition of 10 µM Zn²⁺ caused a marked inhibition of uptake following treatment with 0.5 mM MTSET in all three mutants, with the largest effect found in E2C I159C/D345A (60% inhibition, p < 0.001, one-way analysis of variance with Newman-Keuls multiple comparison post-hoc test, Fig. 6A) followed by E2C I159C/K264A (45% inhibition, p < 0.001, Fig. 6A) and E2C I159C/Y335A (28% inhibition, p < 0.01, Fig. 6A). This provides evidence that Zn²⁺ alters the conformational equilibrium of these mutants, thereby making the cysteine in position 159 more accessible to MTSET. Interestingly, dopamine protected against the inactivation observed in the presence of Zn²⁺ in E2C I159C/D345A and E2C I159C/K264A (p < 0.001, Newman-Keuls multiple comparison post-hoc test, Fig. 6B) similar to its effect in NET I155C (17). However, in E2C I159C/Y335A we did not see significant protection by dopamine (Fig. 6B).

Cocaine (10 µM) strongly potentiated the inactivation by 0.5 mM MTSET in all three mutants (Fig. 6). In the absence of Zn²⁺, cocaine increased the inactivation of E2C I159C/K264A and E2C I159C/D345A from 10 and 15%, respectively, to 55 and 68%, respectively (p < 0.001, Fig. 6C). The addition of Zn²⁺ did not further change this inactivation (Fig. 6C). In E2C I159C/Y335A, cocaine caused 28% inactivation as compared with no inhibition in the absence of cocaine (p < 0.001), however, in the presence of Zn²⁺, inactivation increased from 30 to 70% (p < 0.001, Fig. 6). These data indicate that cocaine and Zn²⁺ both induce in the mutant transporters a conformational change that substantially increases the reactivity of I159C to MTSET.

To exclude the possibility that MTSET inactivation of E2C I159C/K264A, E2C I159C/Y335A, and E2C I159C/D345A in the presence of Zn²⁺ and/or cocaine is because of exposure of an endogenous cysteine as an indirect result of mutating Ile-159 to cysteine, we mutated Ile-159 also to alanine in the background of E2C and E2C D345A. The resulting mutants (E2C I159A and E2C I159A/D345A) displayed uptake properties similar to E2C I159C and E2C I159C/D345A (data not shown); furthermore, we did not observe any significant MTSET inactivation of E2C I159A (data not shown) or E2C I159A/D345A either in the presence of Zn²⁺ or cocaine (Fig. 6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recently, we demonstrated that mutation of the highly conserved Tyr-335 in the third ICL of hDAT dramatically alters the functional effect of high affinity Zn²⁺ binding to the transporter (20). Moreover, the mutation of Tyr-335 to alanine (Y335A) was accompanied by a substantial loss of affinity for cocaine-like inhibitors (150-fold) rendering the transporter essentially insensitive to modulation by these psychostimulants (20). It has also recently been proposed that Y335A might act as a dominant negative mutation impairing the function of a co-expressed WT transporter (27). In the current study, we sought to identify additional mutants that displayed a similar phenotype as Y335A by mutating 16 selected residues in the predicted intracellular domain of the hDAT. Moreover, we applied the substituted cysteine accessibility method (28) to establish a structural read-out reflecting the conformational state of mutant transporters characterized by a Y335A-like phenotype.

Three of the 16 mutants displayed characteristics similar to that of Y335A. Thus, in K264A, D345A, and D436A both the V_max values and the K_m for [³H]dopamine uptake decreased considerably (Table I). For D345A and D436A these properties are in agreement with earlier observations (29). Similar to our previous finding in Y335A, in the presence of micromolar concentrations of Zn²⁺, the uptake capacity in all three mutants was potentiated (Fig. 2 and Table II). In D345A and K264A, which were characterized in detail, the V_max values were restored to the V_max observed for WT in the presence of Zn²⁺ (Table II). Although the relative potentiation of Y335A was higher than that found in K264K and D345A (Fig. 2), Zn²⁺ was not capable of restoring V_max to WT level in this mutant (Table II and Ref. 20). In both K264A and D345A the apparent affinities for cocaine-like inhibitors (cocaine itself and CFT) and for GBR 12,909 were clearly decreased but by 1 order of magnitude and not 2 orders of magnitude as observed in Y335A (Table II). Nonetheless, given that the qualitative changes in the three mutations are similar, i.e. characterized by potentiation rather than inhibition by Zn²⁺, decreased inhibitor affinity, and increased substrate affinity, we infer that the molecular mechanisms underlying the observed changes are similar.

We envisioned previously that the characteristic phenotype of Y335A was because of disruption of critical intramolecular interactions, which lead to a constitutive change in the distribution between conformational states in the transport cycle (20). This interpretation of the Y335A data introduced a new paradigm in this class of transporters with parallels to the concept underlying constitutively activating mutations in G protein-coupled receptors (30–32). Substantial experimental evidence suggests that such mutations promote the release of intramolecular constraining interactions in the receptor molecule, leading to spontaneous formation of the active receptor state and, thus, a change in the conformational equilibrium between inactive and active receptor conformations (30–32). Importantly, constitutively activating mutations in G protein-coupled receptors have proven to be highly valuable tools for gaining insight into the molecular function of this family of membrane proteins (30–32) as well as for drug development.

To substantiate our hypothesis and thus to assess the conformational state of the transporter we have characterized the accessibility of a cysteine engineered into position 159 in TM 3 of hDAT to the positively charged sulfhydryl-reactive compound MTSET. This position was chosen because Rudnick and co-workers (17) recently provided evidence that the accessibility of MTSET to a cysteine engineered in the corresponding position of the homologous SERT and NET is dependent on whether the transporter assumes an outward facing conformation versus an inward facing conformation. According to the classical alternating access model for the function of this class of transporters, it was hypothesized that this position is part of an extracellular gate and thus accessible in the outward facing conformation when the external gate is open, but buried in the protein structure in the inward facing conformation when the external gate is predicted to be closed (17).

Taken together, the MTSET data from the three mutants (E2C I159C/K264A, E2C I159C/Y335A, and E2C I159C/D345A) are consistent with our predictions. In the absence of Zn²⁺, MTSET did not cause a measurable decrease in [³H]dopamine uptake capacity in these mutants (Fig. 6). This is in accordance with the notion that in the absence of Zn²⁺ the mutant transporters primarily reside in an inward facing conformation with position 159 buried in the protein structure. However, the presence of 10 µM Zn²⁺ enhanced inactivation by MTSET and thus accessibility of I159C to the external medium as reflected by 60% inactivation in D345A, 45% in K264A, and 30% in Y335A (Fig. 6). This suggests that Zn²⁺ is capable of restoring a conformational equilibrium in the three mutants with a larger fraction of transporter molecules stabilized in an outward facing conformation in which position 159 is exposed. Interestingly, in K264A and D345A we observed that dopamine is capable of protecting against this inactivation similar to what was observed previously in NET I155C (17). This is consistent with the prediction that dopamine binding initiates transport, resulting in a higher fraction of transporters in an inward facing conformation in which I159C is inaccessible to MTSET.

Although the MTSET mutant data corresponded to our predictions, this was not the case for the WT. Despite the homology of DAT with NET and SERT, MTSET caused only a minor and statistically insignificant inhibition (10%) in hDAT E2C I159C under conditions in which MTSET inhibited uptake by 60–80% in NET and SERT (17). This was not altered either by the addition of dopamine or cocaine, in clear contrast to NET, in which dopamine protected robustly against MTSET inactivation and cocaine strongly enhanced inactivation by MTSET (17). This suggests a conformational difference between DAT and NET/SERT as well as between DAT and the intracellular DAT mutants, with DAT predominantly assuming a conformation in which position 159 is buried, in contrast to NET, SERT, and the mutants in the presence of Zn²⁺. Given the profound effect on uptake of MTSET derivatization of I179C and I155C in the homologous SERT and NET, respectively, it seems unlikely that I159C in hDAT E2C reacts with MTSET but that this does not result in impairment of uptake. The finding that E2C I159C in the context of Y335A, D345A, and K264A can be profoundly inactivated by MTSET further argues against this possibility. Moreover, it could be argued that regardless of whether MTSET reacts or not with I159C in E2C I159C, the ability of Zn²⁺ and cocaine to enhance MTSET inactivation selectively in E2C I159C/Y335A, E2C I159C/D345A, and E2C I159C/K264A and not in E2C I159C can only be explained by a conformational difference between these mutants and E2C I159C. Similarly, the different response to MTSET in hDAT E2C I159C as compared with the corresponding mutation in SERT and NET must be indicative of conformational differences between the different transporters.

The data shown in Fig. 6 suggest that Zn²⁺ promoted activation of the mutant transporters does not completely restore the conformational equilibrium of the hDAT WT but likely generates a transporter with a conformational equilibrium similar, but not necessarily identical, to NET. A partial reversal by Zn²⁺ is supported directly by the observation that the K_m values for both [³H]dopamine and [³H]norepinephrine are lower in the mutants and more "NET-like" even in the presence of Zn²⁺ (Table II). In addition, the V_max ratio between that of [³H]dopamine and [³H]norepinephrine tended to be more NET-like than "DAT-like" in the mutants also in the presence of Zn²⁺ (Table IV). The apparent affinities for inhibitors are also only partially "normalized" by Zn²⁺ in the three mutants (Table II).

From a kinetic perspective one explanation for the data could be that when substrate is absent a larger fraction of the NET transporter molecules than of the DAT transporter molecules reside in the outward facing conformation with the putative external gate open and with position 155 (NET)/159 (DAT) exposed. The DAT might reside relatively briefly in this conformation no matter whether Zn²⁺ is present or not. As a consequence the likelihood of MTSET reacting with I159C in the DAT would be substantially lower than in the NET. In the DAT mutants (K264A, Y335A, and D345A), the chances that the transporter assumes the outward facing conformation in the absence of Zn²⁺ may be even further reduced. In the presence of Zn²⁺, however, the fraction might increase substantially, resulting in an equilibrium similar but not necessarily identical to that of the hNET. In other words, the Zn²⁺-bound mutant, like NET, resides longer in the outward facing conformation with the external gate open, in contrast to DAT WT, which is less likely to assume this conformational state. An alternative explanation would be the existence of structural intermediates between the outward and inward facing conformations. It could be envisioned, for example, that I159C selectively is accessible in such an intermediary conformation in which the DAT only briefly resides in contrast to NET and SERT, which may reside in such a conformation for longer. In case of the mutants, Zn²⁺ might promote a conformational distribution in which such a structural intermediate is more populated than in the DAT WT, resulting in the NET-accessibility pattern.

Although cocaine has a profound effect on the accessibility of I159C in all three mutants, both in the absence and presence of Zn²⁺, cocaine does not promote accessibility of I159C in the E2C background. This suggests that the cocaine-bound state of DAT WT is distinct from that of the mutant transporters. Moreover, it suggests that the cocaine-bound state of NET is different from that of DAT and possibly more similar to that of the mutants, because MTSET dramatically enhanced accessibility of the corresponding I155C in this transporter (17). Importantly, the profound effect of cocaine on MTSET accessibility in the mutants also lends further support to the growing realization that cocaine is not a "silent" inhibitor but rather promotes significant conformational changes in the monoamine transporters (15, 17, 33). In this context, loss of affinity in K264A, Y335A, and D345A for cocaine and cocaine-like compounds as well as for structurally distinct inhibitors such as GBR 12,909 is highly interesting. The loss of affinity for inhibitors clearly demonstrates a key role of the mutated residues in maintaining the structural integrity of the inhibitor binding pocket. We think these results most likely are not indicative of a direct involvement of the mutant residues in inhibitor binding, but rather a consequence of an altered conformational equilibrium, in which the transporter more rarely assumes the high affinity binding state for cocaine and other inhibitors. Hence, binding of the inhibitors requires a substantial change in conformational distribution and thus more energy in comparison to binding to the WT, as directly reflected in the lower apparent affinities. This interpretation of our data further underlines the necessity of being extremely cautious when interpreting the growing body of mutagenesis data showing changes in apparent affinities for cocaine and other inhibitors (34–37). Thus, inferences about direct interactions must be supported by other approaches, such as photoaffinity labeling (38, 39).

Summarized, we have identified additional residues, which upon mutation display phenotypic characteristics similar to what we have previously observed upon mutating Tyr-335 (20). We propose that these characteristics are the result of disrupting critical intramolecular interactions leading to an altered conformational equilibrium. Interestingly, the three residues identified (Lys-264, Asp-345, and Asp-436) are either positively or negatively charged, suggesting that intramolecular salt bridges could be important. Notably, for certain G protein-coupled receptors it is known that intramolecular salt bridges play a key role in regulating the equilibrium between inactive and active receptor states (40, 41). A similar scenario could occur in transporters such as DAT, i.e. the transport cycle might be controlled by continuous disruption and reformation of intramolecular salt bridges involving residues such as Lys-264, Asp-345, and Asp-436. We attempted to test specifically whether Lys-264 and Asp-345 or Lys-264 and Asp-436 formed a salt bridge by exploring the possibility of interchanging their positions (K264D/D345K and K264D/D436K) but unfortunately this resulted in non-functional transporters (data not shown). It is, furthermore, intriguing that the mutants with inverted Zn²⁺ sensitivity all are situated in the three "central" short ICLs of the transporter, ICL 2, 3, and 4 (Fig. 1), especially because the accessibility of MTS reagents to cysteines in these loops in hDAT and in SERT have been shown to be sensitive to the presence of substrate and/or inhibitors (15, 16, 18, 19). It is attractive to suggest that these loops form part of a conformationally active intracellular gating domain that controls access of the substrate to the intracellular milieu and in doing so plays a key role in regulating the overall conformational equilibrium of the transport cycle. Residues critical for maintaining this equilibrium of the transport cycle might, however, also be found in other parts of the transporter. Recent electrophysiological analysis of two mutations at the extracellular end of TM7 in the -aminobutyric acid transporter-1 indicate the presence of major changes in distribution of conformational states in the translocation cycle and accordingly that residues on the extracellular face of the transporter also can be critical for regulating the conformational distribution (42).

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Grants P01 DA 12408 (to U. G. and J. A. J.), the Lundbeck Foundation (to U. G.), the Novo Nordic Foundation (to U. G.), National Institutes of Health Grants DA 11495 and MH 57324 (to J. A. J.), and the Lebovitz Fund (to J. A. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Molecular Neuropharmacology Group, Dept. of Pharmacology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark. Tel.: 45-3532-7548; Fax: 45-3532-7610; E-mail: gether{at}neuropharm.ku.dk.

¹ The abbreviations used are: DAT, dopamine transporter; hDAT, human dopamine transporter; NET, norepinephrine transporter; SERT, serotonin transporter; CFT, 2-carbomethoxy-3-(4-fluorophenyl)tropane; MTS, methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl]-methanethiosulfonate; TM, transmembrane segment; WT, wild type; ICL, intracellular loop.

² L. Shi and J. A. Javitch, manuscript in preparation.

	ACKNOWLEDGMENTS

 
We thank Dr. Harald Sitte for critical reading of the manuscript and Dorthe Vang Larsen for excellent technical assistance.

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