Cationic interactions at the human dopamine transporter reveal binding conformations for dopamine distinguishable from those for the cocaine analog 2 alpha-carbomethoxy-3 alpha-(4-fluorophenyl)tropane.
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.
Chen
Nianhang
N
Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine, Peoria, Illinois 61656, USA. nhc@uic.edu
Sun
LingLing
L
Reith
Maarten E A
ME
D001667:Binding, Competitive / Q000187:drug effects; D002412:Cations / Q000378:metabolism*; D002460:Cell Line; D003042:Cocaine / Q000031:analogs & derivatives* / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins; D004347:Drug Interactions; D006801:Humans; D008094:Lithium / Q000494:pharmacology; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D011188:Potassium / Q000494:pharmacology; D011522:Protons; D012964:Sodium / Q000494:pharmacology
D002412:Cations; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011522:Protons; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D008094:Lithium; D012964:Sodium; D003042:Cocaine; D011188:Potassium; D004298:Dopamine