It has been suggested that cocaine and mazindol bind to separate sites on the dopamine transporter. In the present study, we address this issue by examining the inhibition by mazindol of the binding of [3H]WIN 35,428 ([3H]2beta-carbomethyoxy-3beta-(4-fluorophenyl)-tropane), a phenyltropane analog of cocaine, and the inhibition by WIN 35,428 of [3H]mazindol binding to the cloned human dopamine transporter expressed in C6 glioma cells. The design involved the construction of inhibition curves at six widely different radioligand levels, enabling the distinction between the nonlinear hyperbolic competition (i.e., negative allosteric) model and the competitive (i.e., mutually exclusive binding) model. Nonlinear computer curve-fitting analysis indicated no difference in the goodness of fit between the two models; the negative allosteric model indicated an extremely high allosteric constant of approximately > or = 100, which practically equates to the competitive model. The present results suggest that complex interactions reported between cocaine and mazindol in inhibiting dopamine transport are beyond the level of ligand recognition.
Abstract : Although much is known about the effects of Na + , K + , and Cl ‐ on the functional activity of the neuronal dopamine transporter, little information is available on their role in the initial event in dopamine uptake, i.e., the recognition step. This was addressed here by studying the inhibition by dopamine of the binding of [ 3 H]WIN 35,428 {2β‐carbomethoxy‐3β‐(4‐fluorophenyl)[ 3 H]tropane}, a phenyltropane analogue of cocaine, to the cloned human dopamine transporter expressed in HEK‐293 cells. The decrease in the affinity of dopamine (or WIN 35,428) binding affinity with increasing [K + ] could be fitted to a competitive model involving an inhibitory cation site (1) overlapping with the dopamine (or WIN 35,428) domain. The K + IC 50 for inhibiting dopamine or WIN 35,428 binding increased linearly with [Na + ], indicating a K D,Na+ of 30‐44 m M and a K D,K+ of 13‐16 m M for this cation site. A second Na + site (2), distal from the WIN 35,428 domain but linked by positive allosterism, was indicated by model fitting of the WIN 35,428 binding affinities as a function of [Na + ]. No strong evidence for this second site was obtained for dopamine binding in the absence or presence of low (20 m M ) Cl ‐ and could not be acquired for high [Cl ‐ ] because of the lack of a suitable substitute ion for Na + . The K D but not B max of [ 3 H]WIN 35,428 binding increased as a function of the [K + ]/[Na + ] ratio regardless of total [Cl ‐ ] or ion tonicity. A similar plot was obtained for the K i of dopamine binding, with Cl ‐ at ≥ 140 m M decreasing the K i . At 290 m M Cl ‐ and 300 m M Na + the potency of K + in inhibiting dopamine binding was enhanced as compared with the absence of Cl ‐ in contrast to the lack of effect of Cl ‐ up to 140 m M (Na + up to 150 m M ). The results indicate that Cl ‐ at its extracellular level enhances dopamine binding through a mechanism not involving site 1. The observed correspondence between the WIN 35,428 and dopamine domains in their inclusion of the inhibitory cation site explains why many of the previously reported interrelated effects of Na + and K + on the binding site of radiolabeled blockers to the dopamine transporter are applicable to dopamine uptake in which dopamine recognition is the first step.
The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and acidic residues that are completely or highly conserved among Na + /Cl − ‐dependent transporters. We have explored the roles of these residues using non‐conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184L) lacked plasma membrane immunostaining and were not functional. Both DA uptake and cocaine analog (i.e. 2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative selectivity of the hDAT for cocaine analogs and DA by 10–24‐fold. In particular, mutation of D345 in the third intracellular loop still allowed considerable [ 3 H]DA uptake, but caused undetectable [ 3 H]CFT binding. Upon anti‐C‐terminal‐hDAT immunoblotting, D345N appeared as broad bands of 66–97 kDa, but this band could not be photoaffinity labeled with cocaine analog [ 125 I]‐3β‐( p ‐chlorophenyl)tropane‐2β‐carboxylic acid ([ 125 I]RTI‐82). Unexpectedly, in this mutant, cocaine‐like drugs remained potent inhibitors of [ 3 H]DA uptake. CFT solely raised the K m of [ 3 H]DA uptake in wild‐type hDAT, but increased K m and decreased V max in D345N, suggesting different mechanisms of inhibition. The data taken together indicate that mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding of cocaine to the transporter may not be the only way by which cocaine analogs inhibit DA uptake.
Abstract: Little information is available on the role of Na + , K + , and Cl ‐ in the initial event of uptake of substrates by the dopamine transporter, i.e., the recognition step. In this study, substrate recognition was studied via the inhibition of binding of [ 3 H]WIN 35,428 [2β‐carbomethoxy‐3β‐(4‐fluorophenyl)[ 3 H]tropane], a cocaine analogue, to the human dopamine transporter in human embryonic kidney 293 cells. D‐Amphetamine was the most potent inhibitor, followed by p ‐tyramine and, finally, dl ‐octopamine; respective affinities at 150 m M Na + and 140 m M Cl ‐ were 5.5, 26, and 220 μ M . For each substrate, the decrease in the affinity with increasing [K + ] could be fitted to a competitive model involving the same inhibitory cation site (site 1) overlapping with the substrate domain as reported by us previously for dopamine. K + binds to this site with an apparent affinity, averaged across substrates, of 9, 24, 66, 99, and 134 m M at 2, 10, 60, 150, and 300 m M Na + , respectively. In general, increasing [Na + ] attenuated the inhibitory effect of K + in a manner that deviated from linearity, which could be modeled by a distal site for Na + , linked to site 1 by negative allosterism. The presence of Cl ‐ did not affect the binding of K + to site 1. Models assuming low binding of substrate in the absence of Na + did not provide fits as good as models in which substrate binds in the absence of Na + with appreciable affinity. The binding of dl ‐octopamine and p ‐tyramine was strongly inhibited by Na + , and stimulated by Cl ‐ only at high [Na + ] (300 m M ), consonant with a stimulatory action of Cl ‐ occurring through Na + disinhibition.
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