Which form of dopamine is the substrate for the human dopamine transporter: The cationic or the uncharged species?

The question of which is the active form of dopamine for the neuronal dopam ine transporter is addressed in HEK-293 cells expressing the human dopamine transporter. The K-m value for [H-3]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 [H-3]2 beta-carbo methoxy-3 beta- (4-fluorophenyl) tropane binding displayed an identical pH dependence, suggesting that changes in uptake result from changes in dopami ne recognition. Dopamine can exist in the anionic, neutral, cationic, or zw itterionic form, and the contribution of each form was calculated. The cont ribution of the anion is extremely low (less than or equal to 0.1%), and it s pH dependence differs radically from that of dopamine binding, The increa se in the neutral form upon raising the pH can model the results only when the pK(a1) (equilibrium neutral-charged) is set to a much lower value (6.8) than reported for dopamine in solution (8.86). The sum of cationic and zwi tterionic dopamine concentrations remained constant over the entire pH rang e studied. These forms are the likely transporter substrates with pH-depend ent changes occurring in their interaction with the transporter. The bindin g of dopamine, a hydroxylated phenylethylamine derivative, displays the sam e pH dependence as guanethidine, a heptamethyleniminoethylguanidine derivat ive fully protonated under our conditions. An ionizable residue in the tran sporter could be involved that does not interact with or impact the binding of bretylium, a quaternary ammonium phenylmethylamine derivative that is a lways positively charged and shows only a minor reduction in K-I upon incre asing pH.