The role of zinc ions in reverse transport mediated by monoamine transporters.
The human dopamine transporter (hDAT) contains an endogenous high affinity Zn2+ binding site with three coordinating residues on its extracellular face (His193, His375, and Glu396). Upon binding to this site, Zn2+ causes inhibition of [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) uptake. We investigated the effect of Zn2+ on outward transport by superfusing hDAT-expressing HEK-293 cells preloaded with [3H]MPP+. Although Zn2+ inhibited uptake, Zn2+ facilitated [3H]MPP+ release induced by amphetamine, MPP+, or K+-induced depolarization specifically at hDAT but not at the human serotonin and the norepinephrine transporter (hNET). Mutation of the Zn2+ coordinating residue His(193) to Lys (the corresponding residue in hNET) eliminated the effect of Zn2+ on efflux. Conversely, the reciprocal mutation (K189H) conferred Zn2+ sensitivity to hNET. The intracellular [3H]MPP+ concentration was varied to generate saturation isotherms; these showed that Zn2+ increased V(max) for efflux (rather than K(M-Efflux-intracellular)). Thus, blockage of inward transport by Zn2+ is not due to a simple inhibition of the transporter turnover rate. The observations provide evidence against the model of facilitated exchange-diffusion and support the concept that inward and outward transport represent discrete operational modes of the transporter. In addition, they indicate a physiological role of Zn2+, because Zn2+ also facilitated transport reversal of DAT in rat striatal slices.
Scholze
Petra
P
Institute of Pharmacology, University of Vienna, Währingerstrasse 13a, A-1090 Vienna, Austria.
Nørregaard
Lene
L
Singer
Ernst A
EA
Freissmuth
Michael
M
Gether
Ulrik
U
Sitte
Harald H
HH
D000662:Amphetamines / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D007477:Ions; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009154:Mutation; D009479:Neuropeptides; D009638:Norepinephrine / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D050492:Vesicular Biogenic Amine Transport Proteins; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
D000662:Amphetamines; D018076:DNA, Complementary; D007477:Ions; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D050492:Vesicular Biogenic Amine Transport Proteins; D015032:Zinc; D011188:Potassium; D004298:Dopamine; D009638:Norepinephrine