Striatal Transporter for Dopamine: Catechol Structure‐Activity Studies and Susceptibility to Chemical Modification

The apparent second‐order association rate constant of dopamine binding to the striatal transporter (∼1 ± 10 6 M −1 s −1 ) as well as the transporter turnover number (∼1.5 s −1 ) was estimated using rotating disk electrode voltammetry to monitor apparent zero trans entry of dopamine into striatal suspensions. The substrate specificity of the transporter was also assessed using catechol derivatives. Dopamine and norepinephrine were transported, whereas epinephrine and the acidic metabolites of dopamine were not transported. The metabolite, 3‐meth‐oxytyramine, was transported with a K m seven times greater than and a V max close to that for dopamine. 4‐Methoxytyramine was transported more facilely than the 3‐methoxy derivative. N ‐Alkylation of the amine side chain of dopamine reduced transport dramatically. 4‐Ethylcatechol and 3,4‐dihydroxybenzylamine were transported with velocities 79 and 91 % less than that for dopamine, respectively. The rigid analogue 6,7‐dihydroxy‐1,2,3,4‐tetrahydronaphthalene was transported with a greater velocity than the 5,7‐dihydroxy derivative. Finally, the apparent K m values for 4‐ethylcatechol, 1‐amino‐2‐phenylethane, tyramine, and m ‐tyramine as cosubstrates with dopamine were 1.1, 11, 17, and 2.6 μ M , respectively. Pretreatments of striatal suspensions with chloroethylnorapomorphine, N ‐ethylmaleimide, Hg 2+ , 4,5‐dihydroxy‐4,5‐dioxo‐1 H ‐pyrrolo[2,3‐ f ]quinoline‐2,7,9‐tricarboxylic acid (a redox modulator of receptors in neuronal as well as other tissues), and neuraminidase reduced the velocity of transport of dopamine, whereas N ‐ethoxycarbonyl‐2‐ethoxy‐1,2‐dihydroquinoline had no effect. Thus, the dopamine transporter requires an intact catechol with a primary ethylamine side chain for optimal activity relative to shorter side chain derivatives (side chains longer than two carbons were not tested), the 3‐hydroxyl group of dopamine is the more critical hydroxyl group, and the β rotamer of the extended conformation of dopamine is transported preferentially. The catechol appears to mediate the recognition of the substrate, whereas the amine side chain apparently facilitates the conformational change of the transporter that results in movement of dopamine into or across the membrane. The transporter distinguishes between agents known to block dopamine recognition sites on dopamine receptors? appears to possess a reduction/oxidation modulatory site, and requires sulfhydryl groups and external glycosylation for optimal function.