The inhibitor benztropine was unique in its inability to protect Cys-135. Moreover, whereas cocaine, WIN, mazindol, and dopamine enhanced the reaction of Cys-90 with MTSET, benztropine had no effect on this reaction. These two features combine to give benztropine its weak potency in protecting ligand binding to wild-type DAT from MTSET. These results indicate that different inhibitors of DAT, such as cocaine and benztropine, produce different conformational changes in the transporter. There are differences in the psychomotor stimulant-like effects of these compounds, and it is possible that the different behavioral effects of these DAT inhibitors stem from their different molecular actions on DAT.
The question of which is the active form of dopamine for the neuronal dopamine transporter is addressed in HEK-293 cells expressing the human dopamine transporter. The K m value for [ 3 H]dopamine uptake fell sharply when the pH was increased from 6.0 to 7.4 and then changed less between pH 7.4 and 8.2. The K I for dopamine in inhibiting the cocaine analog [ 3 H]2-carbomethoxy-3-(4-fluorophenyl)tropane binding displayed an identical pH dependence, suggesting that changes in uptake result from changes in dopamine recognition. Dopamine can exist in the anionic, neutral, cationic, or zwitterionic form, and the contribution of each form was calculated. The contribution of the anion is extremely low (<0.1%), and its pH dependence differs radically from that of dopamine binding. The increase in the neutral form upon raising the pH can model the results only when the pK a1 (equilibrium neutralcharged) is set to a much lower value (6.8) than reported for dopamine in solution (8.86). The sum of cationic and zwitterionic dopamine concentrations remained constant over the entire pH range studied. These forms are the likely transporter substrates with pH-dependent changes occurring in their interaction with the transporter. The binding of dopamine, a hydroxylated phenylethylamine derivative, displays the same pH dependence as guanethidine, a heptamethyleniminoethylguanidine derivative fully protonated under our conditions. An ionizable residue in the transporter could be involved that does not interact with or impact the binding of bretylium, a quaternary ammonium phenylmethylamine derivative that is always positively charged and shows only a minor reduction in K I upon increasing pH.The dopamine (DA) 1 transporter (DAT) in neuronal plasma membranes clears DA from the (extra)synaptic space (1-3) by an active uptake process with co-transport of Na ϩ and Cl Ϫ (for recent reviews see Refs. 4 and 5) but probably not countertransport of K ϩ (6). In calculating the overall stoichiometry of the neuronal DA uptake process as 2:1:1 for Na ϩ :Cl Ϫ :DA, the authors have combined the evidence for co-transport of two Na ϩ ions and one Cl Ϫ ion per DA molecule with the assumption that the cationic form of DA is the substrate for uptake (7-9). DA has an amino group that can accept a proton and a phenolic hydroxyl group that can donate a proton; the second phenolic group has a pK a value greater than 12. Therefore, except at extremely basic pH values where both hydroxyl groups can be dissociated, DA can exist as a cation ( ϩ H 3 NDOH, with D for DA skeleton), a zwitterion ( ϩ H 3 NDO Ϫ ), a neutral form (H 2 NDOH), or an anion (H 2 NDO Ϫ ). With pK a1 and pK a2 values of 8.86 and 10.5, respectively (10 -12) (see "Experimental Procedures"), it can be calculated that at physiological pH, DA exists mostly as a cation, which has prompted the assumption that this is the active form for transport (7-9). Indeed, in the analogous cases of neuronal uptake of serotonin and norepinephrine, strong evidence for translocation of the cationic form has ...
The present study elucidated the role of aspartate 345, a residue conserved in the third intracellular loop of all Na ؉ /Cl ؊ -dependent neurotransmitter transporters, in conformational changes of the dopamine (DA) transporter. Asparagine substitution (D345N) resulted in near normal transporter expression on the cell surface but caused extremely low V max and K m values for DA uptake, converted the inhibitory effect of Zn 2؉ on DA uptake to a stimulatory one, and eliminated reverse transport. The cocaine-like inhibitor 2-carbomethoxy-3-(4-fluorophenyl)tropane or the selective DA transporter inhibitor GBR12935 bound to D345N with a normal affinity and still inhibited DA uptake potently. However, the mutation reduced the binding capacity of the surface transporter for these two inhibitors by 90% or more. Moreover, the binding activity of D345N can be significantly improved by Zn 2؉ but not by Na ؉ . These results are consistent with a defect in reorientation of the substrate-binding site to the extracellular side, leading to a loss of the outward-facing conformational state where external DA binds to initiate uptake and the inhibitors bind to initiate uptake inhibition. Alanine or glutamate substitution produced a similar phenotype, suggesting that both the negative charge and the residue volume at position 345 are vital. Furthermore, in intact cells, cocaine potentiated the reaction of the membrane-impermeant sulfhydryl reagent methanethiosulfonate ethyltrimethylammonium with the extracellularly located endogenous cysteines of D345N but not those of wild type, and this potentiation was blocked upon K ؉ substitution for Na ؉ . Thus, cocaine binding to D345N likely induces a different and Na ؉ -dependent conformational change, which may contribute to its Na ؉ -dependent uptake inhibitory activity.
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