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Delineation of an endogenous zinc‐binding site in the human dopamine transporter
Author(s) -
Norregaard Lene,
Frederiksen Dorte,
Nielsen Elsebet Ø.,
Gether Ulrik
Publication year - 1998
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1093/emboj/17.15.4266
Subject(s) - biology , human physiology , neurochemistry , physiology , neuroscience , neurology , endocrinology
The molecular basis for substrate translocation in the Na + /Cl − ‐dependent neurotransmitter transporters remains elusive. Here we report novel insight into the translocation mechanism by delineation of an endogenous Zn 2+ ‐binding site in the human dopamine transporter (hDAT). In micromolar concentrations, Zn 2+ was found to act as a potent, non‐competitive blocker of dopamine uptake in COS cells expressing hDAT. In contrast, binding of the cocaine analogue, WIN 35,428, was markedly potentiated by Zn 2+ . Surprisingly, these effects were not observed in the closely related human norepinephrine transporter (hNET). A single non‐conserved histidine residue (His193) in the large second extracellular loop (ECL2) of hDAT was discovered to be responsible for this difference. Thus, Zn 2+ modulation could be conveyed to hNET by mutational transfer of only this residue. His375 conserved between hDAT and hNET, present in the fourth extracellular loop (ECL4) at the top of transmembrane segment VII, was identified as a second major coordinate for Zn 2+ binding. These data provide evidence for spatial proximity between His193 and His375 in hDAT, representing the first experimentally demonstrated proximity relationship in an Na + /Cl − ‐dependent transporter. Since Zn 2+ did not prevent dopamine binding, but inhibited dopamine translocation, our data suggest that by constraining movements of ECL2 and ECL4, Zn 2+ can restrict a conformational change critical for the transport process.