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
- 6 June 2002
- journal article
- Published by Wiley in Journal of Neurochemistry
- Vol. 81 (6), 1383-1393
- https://doi.org/10.1046/j.1471-4159.2002.00941.x
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 K (i), but not CFT K (d). 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.Keywords
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