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 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 [2beta-carbomethoxy-3beta-(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 by p-tyramine and, finally, dl-octopamine; respective affinities at 150 mM Na+ and 140 mM Cl- were 5.5, 26, and 220 microM. 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 mM at 2, 10, 60, 150, and 300 mM Na+, 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 of dl-octopamine and p-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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Department of Biology, Illinois State University, Normal, USA.
Reith
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D018759:Adrenergic Uptake Inhibitors / Q000378:metabolism*; D000316:Adrenergic alpha-Agonists / Q000378:metabolism; D000494:Allosteric Regulation; D000661:Amphetamine / Q000378:metabolism*; D001665:Binding Sites / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D003042:Cocaine / Q000031:analogs & derivatives / Q000494:pharmacology; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000166:cytology; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008956:Models, Chemical; D009419:Nerve Tissue Proteins; D009655:Octopamine / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D014316:Tritium; D014439:Tyramine / Q000378:metabolism
D018759:Adrenergic Uptake Inhibitors; D000316:Adrenergic alpha-Agonists; D002352:Carrier Proteins; D002712:Chlorides; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D014316:Tritium; D009655:Octopamine; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D012964:Sodium; D000661:Amphetamine; D003042:Cocaine; D011188:Potassium; D014439:Tyramine