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

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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 byp‐tyramine and, finally,dl‐octopamine; respective affinities at 150 mMNa+and 140 mMCl‐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 mMat 2, 10, 60, 150, and 300 mMNa+, 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 ofdl‐octopamine andp‐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.

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