Site-directed mutations near transmembrane domain 1 alter conformation and function of norepinephrine and dopamine transporters.

The human dopamine and norepinephrine transporters (hDAT and hNET, respectively) control neurotransmitter levels within the synaptic cleft and are the site of action for amphetamine (AMPH) and cocaine. We investigated the role of a threonine residue within the highly conserved and putative phosphorylation sequence RETW, located just before transmembrane domain 1, in regulating hNET and hDAT function. The Thr residue was mutated to either alanine or aspartate. Similar to the inward facing T62D-hDAT, T58D-hNET demonstrated reduced [(3)H]DA uptake but enhanced basal DA efflux compared with hNET with no further effect of AMPH. The mutations had profound effects on substrate function and binding. The potency of substrates to inhibit [(3)H]DA uptake and compete with radioligand binding was increased in T→A and/or T→D mutants. Substrates, but not inhibitors, demonstrated temperature-sensitive effects of binding. Neither the functional nor the binding potency for hNET blockers was altered from wild type in hNET mutants. There was, however, a significant reduction in potency for cocaine and benztropine to inhibit [(3)H]DA uptake in T62D-hDAT compared with hDAT. The potency of these drugs to inhibit (3)H-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane-1,5-napthalenedisulfonate (WIN35,428) binding was not increased, demonstrating a discordance between functional and binding site effects. Taken together, these results concur with the notion that the T→D mutation in RETW alters the preferred conformation of both hNET and hDAT to favor one that is more inward facing. Although substrate activity and binding are primarily altered in this conformation, the function of inhibitors with distinct structural characteristics may also be affected.