Which Form of Dopamine Is the Substrate for the Human Dopamine Transporter: the Cationic or the Uncharged Species?

Abstract

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 ...

Is Na + required for the binding of dopamine, amphetamine, tyramine, and octopamine to the human dopamine transporter?

“…It was observed in the present experiments that lowering the pH from 7.4 to 7.0 generally affected the binding of [ 3 H]WIN 35,428 more than that of the substrates, in agreement with the same trend seen in our previous work with WIN 35,428 and DA (Berfield et al 1999). The present results also indicated a somewhat greater effect of lowering pH on [ 3 H]WIN 35,428 but not DA binding at 10 mM Na + than at 60-150 mM Na + , indicating the possibility that Na + counteracts H + in the case of WIN 35,428.…”

First Experimental Evidence of Dopamine Interactions with Negatively Charged Model Biomembranes

“…33,34 Charged DA is a hydrophilic species 37 and should interact with the outer layer of lipid membrane, as it was generally predicted by Cantor 4 and confirmed by MD simulations. 31 For such solutes C L = γC L 0 , where γ is the asymmetry parameter.…”

“…37 Recent computational studies 31 indicate that the lipid headgroup region is the most probable site for the localization of DA and its precursor, L-3,4-dihydroxyphenylalanine (L-DOPA). Basing on these results, we suppose that DA/membrane interactions will depend on structure and charge of lipid headgroups.…”

Selective inhibition of monoamine neurotransmitter transporters by synthetic local anesthetics

“…Lowering pH in the incubation solution caused a decrease in [ 3 H]DA uptake, probably due to an increase in K m , as demonstrated in HEK-293 cells expressing the human DAT (Berfield et al 1999;Fig. 3).…”