Interaction of Na+, K+, and Cl- with the binding of amphetamine, octopamine, and tyramine to the human dopamine transporter.

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 [2beta-carbomethoxy-3beta-(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 microM. 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.