Cationic interactions at the human dopamine transporter reveal binding conformations for dopamine distinguishable from those for the cocaine analog 2α‐carbomethoxy‐3α‐(4‐fluorophenyl)tropane

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Abstract
In membrane preparations, CFT, a phenyltropane cocaine analog, and dopamine (DA) interact with the recombinant human dopamine transporter (hDAT) in Na+‐free medium. Na+ markedly increased the transporter's affinity for CFT, but had little or no effect on DA potency for inhibiting CFT binding. Raising [Na+] from 20 to 155 mm reduced Li+‐induced increase in DA Ki, but not CFT Kd. The presence of 155 mm Na+ enhanced the tolerance to low pH of CFT Kd but not DA Ki. Leucine substitution for tryptophan 84 (W84L) in transmembrane domain (TM) 1 or asparagine substitution for aspartate 313 (D313N) in TM 6 did not or only modestly enhance the affinity of Na+‐independent CFT binding, and retained the near normal ability of DA, Li+, K+, or H+ to inhibit this binding. However, the mutations significantly enhanced the Na+ stimulation of CFT binding as well as the Na+ antagonism against Li+ and H+ inhibition of CFT binding. In contrast, the mutations neither changed the Na+‐insensitive feature of DA Ki nor enhanced the Na+ protection of DA Ki against Li+'s inhibitory effect, though they caused Na+ protection of DA Ki against H+'s inhibitory action. These results are consistent with the existence of binding conformations for DA that are distinguishable from those for CFT, and with a differential association of cation interactions with DA and CFT binding. The mutations likely alter Na+‐bound state(s) of hDAT, preferentially strengthening the positive allosteric coupling between Na+ and CFT binding, and reducing the impact of Li+ or H+ on the CFT binding.

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