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An N-terminal threonine mutation produces an efflux-favorable, sodium-primed conformation of the human dopamine transporter.
Fraser R, Chen Y, Guptaroy B, Luderman KD, Stokes SL, Beg A, DeFelice LJ, Gnegy ME.
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The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr62 revealed T62D-human (h)DAT partitions in a predominately efflux-preferring conformation. Compared with wild-type (WT), T62D-hDAT exhibits reduced [(3)H]DA uptake and enhanced baseline DA efflux, whereas T62A-hDAT and WT-hDAT function in an influx-preferring conformation. We now interrogate the basis of the mutants' altered function with respect to membrane conductance and Na(+) sensitivity. The hDAT constructs were expressed in Xenopus oocytes to investigate if heightened membrane potential would explain the efflux characteristics of T62D-hDAT. In the absence of substrate, all constructs displayed identical resting membrane potentials. Substrate-induced inward currents were present in oocytes expressing WT- and T62A-hDAT but not T62D-hDAT, suggesting equal bidirectional ion flow through T62D-hDAT. Utilization of the fluorescent DAT substrate ASP(+) [4-(4-(dimethylamino)styryl)-N-methylpyridinium] revealed that T62D-hDAT accumulates substrate in human embryonic kidney (HEK)-293 cells when the substrate is not subject to efflux. Extracellular sodium (Na(+) e) replacement was used to evaluate sodium gradient requirements for DAT transport functions. The EC50 for Na(+) e stimulation of [(3)H]DA uptake was identical in all constructs expressed in HEK-293 cells. As expected, decreasing [Na(+)]e stimulated [(3)H]DA efflux in WT- and T62A-hDAT cells. Conversely, the elevated [(3)H]DA efflux in T62D-hDAT cells was independent of Na(+) e and commensurate with [(3)H]DA efflux attained in WT-hDAT cells, either by removal of Na(+) e or by application of amphetamine. We conclude that T62D-hDAT represents an efflux-willing, Na(+)-primed orientation-possibly representing an experimental model of the conformational impact of amphetamine exposure to hDAT.
Carvelli,
Dopamine transporters depolarize neurons by a channel mechanism.
2004, Pubmed
Carvelli,
Dopamine transporters depolarize neurons by a channel mechanism.
2004,
Pubmed Carvelli,
Dopamine transporter/syntaxin 1A interactions regulate transporter channel activity and dopaminergic synaptic transmission.
2008,
Pubmed Chen,
Aspartate 345 of the dopamine transporter is critical for conformational changes in substrate translocation and cocaine binding.
2004,
Pubmed Chen,
Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6.
2004,
Pubmed DeFelice,
Transporters as channels.
2007,
Pubmed Erreger,
Currents in response to rapid concentration jumps of amphetamine uncover novel aspects of human dopamine transporter function.
2008,
Pubmed Foster,
Dopamine transporters are phosphorylated on N-terminal serines in rat striatum.
2002,
Pubmed Foster,
Dopamine transporter phosphorylation site threonine 53 regulates substrate reuptake and amphetamine-stimulated efflux.
2012,
Pubmed Guptaroy,
Site-directed mutations near transmembrane domain 1 alter conformation and function of norepinephrine and dopamine transporters.
2011,
Pubmed Guptaroy,
A juxtamembrane mutation in the N terminus of the dopamine transporter induces preference for an inward-facing conformation.
2009,
Pubmed Hahn,
The functional impact of SLC6 transporter genetic variation.
2007,
Pubmed Hamilton,
De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.
2013,
Pubmed Ingram,
Dopamine transporter-mediated conductances increase excitability of midbrain dopamine neurons.
2002,
Pubmed Jardetzky,
Simple allosteric model for membrane pumps.
1966,
Pubmed Khoshbouei,
N-terminal phosphorylation of the dopamine transporter is required for amphetamine-induced efflux.
2004,
Pubmed Khoshbouei,
Amphetamine-induced dopamine efflux. A voltage-sensitive and intracellular Na+-dependent mechanism.
2003,
Pubmed Kniazeff,
An intracellular interaction network regulates conformational transitions in the dopamine transporter.
2008,
Pubmed Krishnamurthy,
X-ray structures of LeuT in substrate-free outward-open and apo inward-open states.
2012,
Pubmed Levi,
Carrier-mediated release of neurotransmitters.
1993,
Pubmed Leviel,
Dopamine release mediated by the dopamine transporter, facts and consequences.
2011,
Pubmed Liang,
Evidence for carrier-mediated efflux of dopamine from corpus striatum.
1982,
Pubmed Liang,
Interaction of catechol and non-catechol substrates with externally or internally facing dopamine transporters.
2009,
Pubmed Loland,
Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding.
2004,
Pubmed Loland,
Generation of an activating Zn(2+) switch in the dopamine transporter: mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle.
2002,
Pubmed Mazei-Robison,
Anomalous dopamine release associated with a human dopamine transporter coding variant.
2008,
Pubmed Meinild,
Zinc potentiates an uncoupled anion conductance associated with the dopamine transporter.
2004,
Pubmed
,
Xenbase Pifl,
Ion dependence of carrier-mediated release in dopamine or norepinephrine transporter-transfected cells questions the hypothesis of facilitated exchange diffusion.
1999,
Pubmed Pifl,
Induction by low Na+ or Cl- of cocaine sensitive carrier-mediated efflux of amines from cells transfected with the cloned human catecholamine transporters.
1997,
Pubmed Raiteri,
Dopamine can be released by two mechanisms differentially affected by the dopamine transport inhibitor nomifensine.
1979,
Pubmed Rudnick,
Ion-coupled neurotransmitter transport: thermodynamic vs. kinetic determinations of stoichiometry.
1998,
Pubmed Schwartz,
Binding and transport in norepinephrine transporters. Real-time, spatially resolved analysis in single cells using a fluorescent substrate.
2003,
Pubmed Shan,
The substrate-driven transition to an inward-facing conformation in the functional mechanism of the dopamine transporter.
2011,
Pubmed Solis,
4-(4-(dimethylamino)phenyl)-1-methylpyridinium (APP+) is a fluorescent substrate for the human serotonin transporter.
2012,
Pubmed
,
Xenbase Sonders,
Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants.
1997,
Pubmed
,
Xenbase Sulzer,
Mechanisms of neurotransmitter release by amphetamines: a review.
2005,
Pubmed Vaughan,
Phosphorylation and regulation of psychostimulant-sensitive neurotransmitter transporters.
2004,
Pubmed Yamashita,
Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters.
2005,
Pubmed Zapata,
Regulation of dopamine transporter function and cell surface expression by D3 dopamine receptors.
2007,
Pubmed Zhao,
Ion-controlled conformational dynamics in the outward-open transition from an occluded state of LeuT.
2012,
Pubmed Zhao,
Single-molecule dynamics of gating in a neurotransmitter transporter homologue.
2010,
Pubmed Zhao,
Substrate-modulated gating dynamics in a Na+-coupled neurotransmitter transporter homologue.
2011,
Pubmed Zhen,
Differences in interactions with the dopamine transporter as revealed by diminishment of Na(+) gradient and membrane potential: dopamine versus other substrates.
2005,
Pubmed
