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 init ial event of uptake of substrates by the dopamine transporter, i.e., the re cognition step. In this study, substrate recognition was studied via the in hibition of binding of [H-3]WIN 35,428 [2 beta-carbomethoxy-3 beta-(4-fluor ophenyl)[H-3]tropane], a cocaine analogue, to the human dopamine transporte r in human embryonic kidney 243 cells. D-Amphetamine was the most potent in hibitor, followed by p-tyramine and, finally, dl-octopamine; respective aff inities at 150 mM Na+ and 140 mM Cl- were 5.5, 26, and 220 mu M. For each s ubstrate, 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 dopa mine. K+ binds to this site with an apparent affinity, averaged across subs trates, of 9, 24, 66, 99, and 134 mM at 2, 10, 60, 150, and 300 mu M Na+, r espectively. 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 presenc e of Cl- did not affect the binding of K+ to site 1. Models assuming low bi nding of substrate in the absence of Na+ did not provide fits as good as mo dels in which substrate binds in the absence of Na+ with appreciable affini ty. The binding of dl-octopamine and p-tyramine was strongly inhibited by N a+, and stimulated by Cl- only at high [Na+] (300 mM), consonant with a sti mulatory action of Cl- occurring through Na+ disinhibition.