Site-Directed Mutations near Transmembrane Domain 1 Alter Conformation and Function of Norepinephrine and Dopamine Transporters

Abstract

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](Ϫ)-2-␤-carbomethoxy-3-␤-(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 T3 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.

Mutation of Tyrosine 470 of Human Dopamine Transporter is Critical for HIV-1 Tat-Induced Inhibition of Dopamine Transport and Transporter Conformational Transitions

“…One caveat is that DA efflux data may reflect not only DA moving out of the cell through the transporter, but also non-specific diffusion and reuptake. However, there is evidence (Guptaroy et al, 2009; Guptaroy et al, 2011) that this measurement largely reflects basal DA efflux through DAT because such basal DA efflux is consistent with amphetamine- or voltage-stimulated efflux of intracellular DA in cells expressing hDAT and its mutant. Amphetamine, a substrate for DAT, competitively inhibits DA reuptake and elicits outward transport of DA by reversal of the transporter (Sulzer et al, 2005).…”

“…In contrast, the DA uptake potency of cocaine and GBR12909 is increased in Y470H-hDAT compared to WT hDAT. While GBR12909 labels the classic DA uptake site in rodent brain, binding to the piperazine acceptor site (Andersen et al, 1987) is affected much less by mutation of hDAT than cocaine (Loland et al, 2002; Guptaroy et al, 2011), consistent with the fact that cocaine preferentially stabilizes the hDAT in the outward-facing conformational state, resulting in a reduction of DA uptake (Reith et al, 2001; Loland et al, 2002). One interpretation of our finding is that Tat allosterically modulates DA transport rather than overlaps DA uptake sites on DAT as previously suggested (Zhu et al, 2011).…”

Reciprocal Phosphorylation and Palmitoylation Control Dopamine Transporter Kinetics

“…These conformations are governed by extracellular and intracellular gates that control substrate access to the permeation pathway (39) and can be further regulated by Zn 2ϩ binding, which stabilizes the outwardly facing form (41,42), and interaction of cytoplasmic domains with syntaxin 1A and other binding partners (37,43,44). The equilibrium between these conformations determines the rate of forward transport and also impacts cocaine analog binding, which is favored by the outwardly facing form, and substrate efflux, which is thought to be favored by the inwardly facing form (45)(46)(47)(48).…”

Mechanisms of dopamine transporter regulation in normal and disease states

“…Great insights on the core structure have been obtained from homology modeling to the bacterial leucine transporter LeuT [1, 10], but far less is known about the cytoplasmic domains, which are not conserved in LeuT and whose structures have not been solved. The N-terminus contains intracellular gate residue Arg60 which stabilizes the outwardly facing form and has been implicated in control of transport by influencing the conformation of TM1a, which is thought to undergo major structural rearrangements during the outward-to-inward transition [11-13]. Less is known about the C-terminal domain or its potential impacts on TM12.…”