Introduction

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The dopamine transporter has been suggested to be the initial target for cocaine in producing its reinforcing and addictive effects (Wise, 1984; Ritz et al., 1987; Carroll et al., 1992). It is widely accepted that blockade of dopamine uptake by cocaine is the result of high-affinity binding of cocaine to the dopamine transporter (Reith et al., 1986; Calligaro and Eldefrawi, 1987; Ritz et al., 1987; Madras et al., 1989). It is not known whether the binding sites for cocaine are unique and whether this accounts for the exceptional abuse potential of cocaine among all compounds that block dopamine uptake (Fahey et al., 1989; Berger et al., 1990; Reith et al., 1992; Carroll et al., 1992; Reith and Selmeci, 1992). Although a competitive model has been advanced for the interaction of cocaine or the close cocaine congener WIN 35,428 and mazindol in equilibrium binding experiments in rodent striatum (Javitch et al., 1984; Calligaro and Eldefrawi, 1987; Reith and Selmeci, 1992; Dersch et al., 1994) or rabbit caudate nucleus (Aloyo et al., 1995), more complex interactions have been observed in monkey caudate putamen (Madras et al., 1989) and rat striatum (Berger et al., 1990). In a recent study, Meiergerd and Schenk (1994) reported that cocaine and mazindol interact with separate but interacting sites in inhibiting dopamine uptake in rat striatal suspensions measured by rotating disk electrode voltammetry.

In the present radioligand-binding study, we examined the issue of whether cocaine and mazindol bind to separate or interacting sites on the dopamine transporter by two new experimental approaches. First, the cloned human dopamine transporter is studied rather than the native rodent transporter because it is known that there are a number of differences between the human and rat dopamine transporter (Giros et al., 1992). Although differences have been reported between cloned and native dopamine transporters within the same species, these have been mostly in the area of substrate interaction or translocation, with generally higher K_d values for [³H]dopamine uptake and K_i values for MPP⁺ (1-methyl-4-phenylpyridinium) or norepinpehrine in inhibiting [³H]dopamine uptake for the clones (Pifl et al., 1993; Giros and Caron, 1993) but comparable inhibitory potencies for a variety of blockers (Pifl et al., 1993; Giros and Caron, 1993; Eshleman et al., 1995). Second, rather than relying on the steepness and completeness of inhibition curves obtained at one level of radioligand (Javitch et al., 1984; Calligaro and Eldefrawi, 1987; Madras et al., 1989; Aloyo et al., 1995) or the change in slope but not abscissa intercept in Scatchard plots (Reith and Selmeci, 1992; Dersch et al., 1994), we now report on experiments involving a series of inhibition curves obtained at radioligand concentrations varying over a wide range, as recommended by Tomlinson and Hnatowich (1988). This design enables the distinction between linear and nonlinear ("hyperbolic") competitive interactions; with one level of radioligand, the latter may be mistaken for simple competitive inhibition. The current experimental design involves two pairs, [³H]WIN 35,428 with mazindol and [³H]mazindol with WIN 35,428, studied under conditions yielding one-site binding for either radioligand.

	Methods

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Models

Hyperbolic competitive model. First, the present inhibition data were analyzed according to the hyperbolic competitive model as defined by Tomlinson and Hnatowich (1988), which is equivalent to the negative allosterism model of Ehlert (1988). In the model, a receptor (R) bears two classes of interacting binding sites: site 1 is the site to which the ligand (L) binds; site 2 binds inhibitor (I) only. I decreases the affinity of R for L by binding to site 2 through an allosteric relationship:

(1)

(2)

(3)

(4)

(5)

Competitive model. It can be easily seen that if   1, the hyperbolic model reverts to competitive inhibition:

(6)

(7)

(8)

C6 glioma cells stable expressing the human dopamine transporter. cDNA encoding the human dopamine transporter was cloned in the laboratory of Dr. Aaron Janowsky (Oregon Health Sciences University, Portland, OR) by screening a human substantia nigra cDNA library with a polymerase chain reaction-amplified probe based on the rat dopamine transporter cDNA sequence (Eshleman et al., 1995). Rat C6 glioma cells were transfected, grown for several passages and frozen in a medium containing 45% Dulbecco's modified Eagle's medium, 5% dimethylsulfoxide and 50% fetal bovine serum for storage in liquid nitrogen. For each experiment, one freezing vial was rapidly thawed and seeded into a 75-cm² flask at a density of ~100,000 cells/cm². When the flask reached confluency (after ~4 days at a density of ~600,000 cells/cm²), the cells were lysed by trypsinization and seeded into four 75-cm² flasks. At ~4 days later, when the flasks reached confluency (~600,00 cells/cm²), the cells were ready for the binding experiment.

Inhibition of [³H]WIN 35,428 binding by mazindol and [³H]mazindol binding by WIN 35,428. Assays were carried out in triplicate in a total volume of 0.2 ml in 1-ml ministrip tubes (Skatron, Sterling, VA). The composition of the assay mixture was 20 µl of radioligand in water, 10 µl of inhibitor in water, 120 µl of assay buffer (33 mM sodium phosphate, pH 7.4, at room temperature) and 50 µl of cell membrane preparations in assay buffer. The incubation was carried out at 0° to 4°C for 2 hr and terminated by filtration with a miniharvesting apparatus (type 11021, Skatron). The detailed procedures of the binding assays were as we previously described (Coffey and Reith, 1994). Nonspecific binding was defined with 100 µM cocaine.

Inhibition of [³H]WIN 35,428 binding by WIN 35,428. Saturation analysis was carried out by adding increasing concentrations (0.5, 1, 2, 5, 10, 20, 40 and 100 nM) of WIN 35,428 to a constant concetration (0.35 nM) of [³H]WIN 35,428. All other procedures were as above.

Data analysis and statistics. IC₅₀ values and pseudo-Hill numbers were computed with the equation of the ALLFIT program of DeLean et al. (1978) entered into the Microsoft ORIGIN curve-fitting and plotting software, which was run with total and nonspecific binding entered as constants. IC₅₀ values of inhibitors (mazindol and WIN 35,428) as a function of varying levels of radioligand ([³H]WIN 35,428 and [³H]mazindol, respectively) were plotted as described above. The dissociation constant of the inhibitor (K₃) obtained from the y axis intercept was compared with the dissociation constant of the same compound when used as a radioligand (K_d = K₁ in above models).

Materials

[³H]WIN 35,428 [lot no. 3141-232; specific activity, 109.3 Ci/mmol; determined by the homologous competition binding method we previously described (Wiener and Reith, 1992)] and [³H]mazindol (lot no. 2824-285; specific activity, 17.0 Ci/mmol) were obtained from DuPont-New England Nuclear (Boston, MA). WIN 35,428 was from Research Biochemicals (Natick, MA). Mazindol was from Sandoz Pharmaceuticals (E. Hanover, NJ). All other chemicals were from Sigma Chemical (St. Louis, MO) or Fisher.

	Results

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Fitting of K₁, K₃ and  in the hyperbolic competitive model. The [Lb]/[Lb]_max values for a representative experiment of inhibition of [³H]WIN 35,428 binding by mazindol at varying [L] are shown in figure 1A, and the mirror experiment with [³H]mazindol and WIN 35,428 is shown in figure 1B. It can be seen that raising [L] resulted in progressively higher [Lb]/[Lb]_max values at a given [I]. Fitting of the data sets of the separate experiments to the hyperbolic competitive model (equation 5) resulted in extremely high values for the allosteric constant  (table 1, top). With all parameters unrestricted, best-fit estimates for  ranged from 106 to 240 for all experiments with either [³H]WIN 35,428 or [³H]mazindol, except for one experiment that gave a value of 1.5 × 10¹³ (table 1, top). There were no statistically significant differences between K_d values that were determined with the compound as the tritiated ligand and K_i values for the same compound as an inhibitor with the alternate compound as the tritiated ligand (table 1, top).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Inhibition of [3H]WIN 35,428 binding by mazindol (A) and [3H]mazindol binding by WIN 35,428 (B). A, , 0.725; , 1.586; , 3.38; , 7.14; , 14.77; and , 30.56 nM [3H]WIN 35,428. B, , 1.1; , 2.31; , 5.24; , 11.11; , 23.43; and *, 49 nM [3H]mazindol. A typical experiment is shown, determined in triplicate, that was carried out three times.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Correlation between the Kd values determined by computer fitting of the data sets to the hyperbolic and apparent competitive model. Each point represents the fitting of one data set. , [3H]Mazindol; , [3H]WIN 35,428 (r = .999, P < .00001).

View this table: [in this window] [in a new window]   TABLE 1 Nonlinear curve fitting to the hyperbolic competitive model of inhibition of [3H]WIN 35,428 binding by mazindol and [3H]mazindol binding by WIN 35,428  Data were analyzed by nonlinear curve fitting to equation 5. With the corresponding compound as the radioligand, Kd = K1 and Ki = K3; with the alternate compound as the radioligand, Kd = K3 and Ki = K1. K values were represented as mean ± S.E.M. for three independent experiments carried out in triplicate;  and mean sum of square values were listed individually. There were no statistically significant differences between Kd and Ki values for the same compound or between  values for the two fitting strategies (Student's t test).

Fitting of K₁ and K₃ in the competitive model. Fitting of the data sets to the competitive model (equation 6) yielded K_d and K_i values virtually indistinguishable from those obtained in the hyperbolic competitive model (compare table 2 with table 1). In fact, the goodness of the fit did not improve in going from the simple to the complex model when tested separately by the F test for each data set (table 2). Furthermore, there was a positive correlation between the nonlinear and linear K_d values found in separate experiments (r = .999, P < .00001) (fig. 2), suggesting that the two models are indistinguishable.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Inhibition of [3H]WIN 35,428 binding by mazindol (A-C) and [3H]mazindol binding by WIN 35,428 (D-F). Inhibition curves were constructed at six different concentrations of radioligand as described in the text; each panel represents one of the six experiments carried out, and IC50 values were plotted against radioligand concentration. Straight lines represent linear least-squares regression fits.

Graphic determination of K₁ and K₃ in the competitive model. The inhibition curves for mazindol in the [³H]WIN 35,428 binding assays and for WIN 35,428 in the [³H]mazindol binding assays shifted to the right with increasing [L] (fig. 1, A and B). The Hill values for inhibition of [³H]WIN 35,428 binding were 0.99 ± 0.03 and those for [³H]mazindol binding were 0.98 ± 0.03. The IC₅₀ values plotted as a function of [L] according to the competitive model (equation 8) fell on a straight line for each data set examined (fig. 3, A-F). There were no statistically significant differences between K_d values that were determined with the compound as the tritiated ligand and K_i values for the same compound as an inhibitor with the alternate compound as the tritiated ligand (table 3). As can be seen in figure 3, there were day-to-day differences in the slopes of the regression lines, which in turn caused variation in the K_d estimates, but, on average, the affinities obtained from this analysis were comparable to those from the hyperbolic competitive analysis (table 1, top).

Fitting of  in the hyperbolic competitive model with K₁ or K₃ as a constant. It was important to make sure that the estimate for allosterism () in the hyperbolic competitive model was not affected in a significant manner by the fitted value of K_d or K_i. Therefore, the data sets were fitted with K_i restrained to the value obtained from the linear regression analysis of the same sets (y intercept in fig. 3, A-F), and again the  values were high, ranging from 132 to 238 except for an extreme outlyer in each group (2.4 × 10¹⁰ and 2.5 × 10¹²) (table 1, bottom). When the K_d value in the hyperbolic competitive model was set to the value arrived at by the competitive graphic analysis, the fitted K_i value for each data set was very close to that deduced from the competitive model (data not shown). Again,  was high, ranging from 94 to 253 with two outlying values (6.2 × 10³ and 1.4 × 10⁹) in the total of six experiments (data not shown).

Saturation analysis of [³H]WIN 35,428 binding. Inhibition of [³H]WIN 35,428 binding by WIN 35,428 was examined under the current conditions (with three independent membrane preparations). Analysis by LIGAND could be performed only with the one-site model in agreement with the linear Scatchard plot (see fig. 4 for a representative experiment).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Saturation analysis of [3H]WIN 35,428 binding. The radioligand was present at 0.35 nM, and increasing concentrations of WIN 35,428 were added up to 100 nM. Nonspecific binding was defined with 100 µM cocaine. The straight line represents the best fit chosen by the LIGAND program, indicating a Kd of 10.4 nM and Bmax of 3.6 pmol/mg of protein. A typical experiment is shown that was carried out three times with independent membrane preparations.

	Discussion

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One-site binding model for the radioligands used. There is general consensus that [³H]mazindol binds to only one site on the dopamine transporter (Javitch et al., 1984; Reith and Selmeci, 1992; Dersch et al., 1994). Indeed, membranes prepared from the present C6 glioma cell system expressing the human dopamine transporter displayed only one component of [³H]mazindol binding as determined in a separate set of recent experiments carried out under slightly different assay conditions (Wu et al., 1997, in press). However, [³H]WIN 35,428 binding has been reported to be heterogeneous for the cloned rat (Boja et al., 1992) and human (Pristupa et al., 1994) dopamine transporter expressed in COS cells. In contrast, one-site binding has been reported for the binding of the closely related [¹²⁵I]RTI-55 ligand to the cloned rat (Gu et al., 1994) and human (Eshleman et al., 1995) dopamine transporter. We examined this issue previously for [³H]WIN 35,428 binding to the current C6 glioma cell preparation with a low concentration of radioligand (0.45 nM) and an extended range of unlabeled WIN 35,428 (0.4-1000 nM) in assays with larger volumes without sucrose as described previously (Reith and Selmeci, 1992); no evidence was found for more than one binding component (Reith et al., 1996). Under the present conditions with sucrose, again only one component was observed (fig. 4). Most likely, the present C6 glioma cell system does not express the low-affinity [³H]WIN 35,428 binding component, which has been suggested to be unrelated to dopamine uptake (Pristupa et al., 1994).

Allosterism in the hyperbolic competitive model vs. competitive binding. The present results show that if there is an allosteric interrelationship between WIN 35,428 and mazindol binding sites, the allosterism is extreme because of the high  values, practically equating the situation to competitive interaction. Indeed, for any given data set, there was no statistically significant difference between the goodness of the fit of the allosteric ( ~ 100) and competitive ( ) model. Furthermore, among different data sets, there was a positive correlation between dissociation constants fitted in the competitive model and those fitted in the allosteric model, indicating that day-to-day variations affected the absolute values of the kinetic constants but not the type of applicable model.

Extrapolation from WIN 35,428 to cocaine binding. There is an ongoing debate as to whether phenyltropane analogs of cocaine interact with the same site that binds cocaine (Madras et al., 1989; Eshleman et al., 1993; Dersch et al., 1994). This is an important issue because [³H]WIN 35,428 or other iodinated phenyltropane analogs of cocaine are commonly used in binding studies instead of [³H]cocaine because of their higher affinity (Madras et al., 1989; Wall et al., 1993; Reith and Coffey, 1993; Gu et al., 1994; Eshleman et al., 1995; Xu et al., 1995). Classic inhibition and saturation studies, although with the limitations pointed out above, have indicated a competitive interaction between WIN 35,428 and cocaine (Reith and Selmeci, 1992; Reith et al., 1992). In addition, studies on structure-activity relationships within the cocaine and phenyltropane family of compounds have shown a generally similar impact on dopamine transporter activity (Carroll et al., 1992). However, as pointed out by the group of Rothman et al. (Dersch et al., 1994), different ligands may be binding to slightly different domains on the dopamine transporter; for instance, nomifensin inhibits [³H]mazindol binding with a K_i value of 24 nM, but [³H]GBR 12935 binding to the dopamine transporter with a K_i value of 236 nM, as determined under the same conditions. In contrast, across the four radioligands studied by the group of Rothman under identical conditions, [³H]mazindol, [³H]GBR 12935, [³H]BTCP and [¹²⁵I]RTI-55, the K_i values for WIN 35,428 were in the same range, (108, 44, 36 and 45 nM, respectively), as were the K_i values for cocaine (767, 660, 689 and 341 nM) (Dersch et al., 1994; Rothman et al., 1994). This suggests that the binding domains for these radioligands, including [³H]mazindol, overlap in comparable way with the binding domains for WIN 35,428 and cocaine. In a recent study, we observed a similar pH sensitivity of the binding of cocaine methiodide and WIN 35,428 to the dopamine transporter, and we ruled out the implication of varying concentrations of protonated and neutral ligand as a function of pH (Xu and Reith, 1996), which is also consonant with the involvement of a common, pH-sensitive domain in cocaine and WIN 35,428 binding. Furthermore, other approaches, such as protection against N-ethylmaleimide-induced alkylation, have not provided evidence in favor of different binding domains for cocaine and WIN 35,428 (Xu et al., 1997) whereas the same technique suggests differences between blocker and substrate domains (Reith et al., 1996; Xu et al., 1997).

Transporter mutants and chimeras. If, indeed, different binding domains are involved in WIN 35,428 and mazindol binding, one would expect certain discrete changes in the coding sequence for the dopamine transporter protein to be ineffective on [³H]WIN 35,428 binding but at the same time reduce the potency of mazindol in inhibiting [³H]WIN 35,428 binding. So far, this type of detailed information has not been obtained in the very recent mutagenesis and chimera studies. Evidence for a role of transmembrane domains, as opposed to the large loop between the third and fourth transmembrane domain, or the amino- and carboxyl-terminal tail in the interaction with compounds has been advanced along with different regions involved in substrate and blocker binding in general (Kitayama et al., 1992; Giros et al., 1994; Buck and Amara, 1994), but further distinctions between certain blockers, such as cocaine and mazindol, must be addressed in future studies.

Effects of cocaine/WIN 35,428 and mazindol on dopamine transport. Although inhibition of [³H]dopamine translocation into striatal synaptosomes by various uptake blockers, including cocaine and mazindol, has been described to be of a competitive nature (Richelson and Pfenning, 1984; Shank et al., 1987; Krueger, 1990), detailed mechanistic studies are lacking. In recent studies using rotating disk voltammetry for the measurement of dopamine uptake into striatal suspensions, the group of Schenk reported a difference in the mode of interaction between dopamine transport and cocaine, involving an uncompetitive action at Na⁺ binding sites (McElvain and Schenk, 1992), and that for mazindol, involving competition for dopamine recognition (Meiergerd and Schenk, 1994). These uptake results taken together may not necessarily present a discrepancy from the binding data because binding studies do not address effects of cocaine beyond the recognition step that affect dopamine translocation. However, if we accept that cocaine inhibits dopamine uptake through a mechanism unrelated to interference with dopamine recognition (i.e., through interaction with Na⁺ binding sites), and if we accept the large body of evidence implicating binding sites for [³H]cocaine and tritiated or iodinated cocaine analog in dopamine uptake inhibition (Ritz et al., 1987; Xu et al., 1995), it follows that the latter binding sites are more closely related to Na⁺ binding than to dopamine recognition. Meiergerd and Schenk postulate mutually interacting sites at which cocaine and mazindol interact to inhibit uptake (Meiergerd and Schenk, 1994), although the mathematical underpinnings regarding the mutual nature have not been made clear. Thus, the dissociation constant for mazindol in binding to the transporter cocaine complex is estimated to be 345 nM (Meiergerd and Schenk, 1994), but the same constant for cocaine in binding to the transporter-mazindol complex is not presented.