IIIIIIInteraction of Na+, K+, and Cl‐ with the Binding of Amphetamine, Octopamine, and Tyramine to the Human Dopamine Transporter

Abstract

Little information is available on the role of Na⁺, K⁺, and Cl^- in the initial event of uptake of substrates by the dopamine transporter, i.e., the recognition step. In this study, substrate recognition was studied via the inhibition of binding of [³H]WIN 35,428 [2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane], a cocaine analogue, to the human dopamine transporter in human embryonic kidney 293 cells. D-Amphetamine was the most potent inhibitor, followed by p-tyramine and, finally, dl-octopamine; respective affinities at 150 mM Na⁺ and 140 mM Cl^- were 5.5, 26, and 220 μM. For each substrate, the decrease in the affinity with increasing [K⁺] could be fitted to a competitive model involving the same inhibitory cation site (site 1) overlapping with the substrate domain as reported by us previously for dopamine. K⁺ binds to this site with an apparent affinity, averaged across substrates, of 9, 24, 66, 99, and 134 mM at 2, 10, 60, 150, and 300 mM Na⁺, respectively. In general, increasing [Na⁺] attenuated the inhibitory effect of K⁺ in a manner that deviated from linearity, which could be modeled by a distal site for Na⁺, linked to site 1 by negative allosterism. The presence of Cl^- did not affect the binding of K⁺ to site 1. Models assuming low binding of substrate in the absence of Na⁺ did not provide fits as good as models in which substrate binds in the absence of Na⁺ with appreciable affinity. The binding of dl-octopamine and p-tyramine was strongly inhibited by Na⁺, and stimulated by Cl^- only at high [Na⁺] (300 mM), consonant with a stimulatory action of Cl^- occurring through Na⁺ disinhibition.