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

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... 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