DELINEATION OF AN ENDOGENOUS ZINC-BINDING SITE IN THE HUMAN DOPAMINE TRANSPORTER

The molecular basis for substrate translocation in the Na+/Cl--depende nt neurotransmitter transporters remains elusive, Here we report novel insight into the translocation mechanism by delineation of an endogen ous Zn2+-binding site in the human dopamine transporter (hDAT), In mic romolar concentrations, Zn2+ was found to act as a potent, non-competi tive blocker of dopamine uptake in COS cells expressing hDAT, In contr ast, binding of the cocaine analogue, WIN 35,428, was markedly potenti ated by Zn2+, Surprisingly, these effects were not observed in the clo sely related human norepinephrine transporter (hNET), A single non-con served histidine residue (His193) in the large second extracellular lo op (ECL2) of hDAT was discovered to be responsible for this difference , Thus, Zn2+ modulation could be conveyed to hNET by mutational transf er of only this residue, His375 conserved between hDAT and hNET, prese nt in the fourth extracellular loop (ECL4) at the top of transmembrane segment VII, was identified as a second major coordinate for Zn2+ bin ding, These data provide evidence for spatial proximity between His193 and His375 in hDAT, representing the first experimentally demonstrate d proximity relationship in an Na+/Cl--dependent transporter, Since Zn 2+ did not prevent dopamine binding, but inhibited dopamine translocat ion, our data suggest that by constraining movements of ECL2 and ECL4, Zn2+ can restrict a conformational change critical for the transport process.