Functional Characterization of N-terminal Threonine Mutations in Human Dopamine and Norepinephrine Transporters.

Human Dopamine Transporter;Human Norepinephrine Transporter;Transporter Conformation and Orientation;Intracellular Gating Network;Conserved RETW Sequence;N-terminal Threonine Mutation;Amphetamine;Sodium Gradient;Pharmacy and Pharmacology;Health Sciences;Pharmacology

Fraser, RheaclareFisher, Stephen K. ;

University of Michigan

关键词: Human Dopamine Transporter;    Human Norepinephrine Transporter;    Transporter Conformation and Orientation;    Intracellular Gating Network;    Conserved RETW Sequence;    N-terminal Threonine Mutation;    Amphetamine;    Sodium Gradient;    Pharmacy and Pharmacology;    Health Sciences;    Pharmacology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/99776/fraserr_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The Na+ and Cl- dependent, human dopamine (DA) and norepinephrine (NE) transporters (hDAT and hNET, respectively) control the duration of DA and NE neurotransmission by taking up extracellular neurotransmitter.Through outward and inward conformational transitions, DAT and NET work as pumps to transport substrates and ions between extracellular and intracellular environments.To study DAT and NET conformations, we mutated the threonine (Thr, T) residue within a conserved RETW sequence, which is juxtaposed to transmembrane domain 1a that is essential for ligand binding.Since the Thr residue is a putative phosphorylation site, it was mutated to alanine (Ala) (DAT, T62A; NET, T58A), or aspartate (Asp) (DAT, T62D; NET, T58D) to mimic a non-phosphorylated and phosphorylated state, respectively.Studies in heterologous HEK293 cells revealed the Thr (T) to Asp (D) mutation profoundly shifts the transporter from a predominately outward to inward orientation that exhibits reduced uptake but enhanced baseline reverse transport of substrate compared to wild type.Thr to Ala (A) mutants mostly functioned like wild type.A consequence of the predominantly inward conformation was an enhanced affinity for all substrates tested in the T to D mutants, with varying impacts of inhibitors.In further investigations, T62D-hDAT demonstrated wild type inward transport and retention of substrate when the substrate was not subject to reverse transport.These data showed that an inward-facing transporter can complete the entire transport cycle, even though it prefers an open inward-gate.Altering the transmembrane Na+ gradient revealed that the Thr to Asp mutation renders the mutant in a conformation mimicking that promoted by the Na+ gradient to elicit reverse transport.This is also the conformation elicited by amphetamine, an abused psychostimulant and transporter substrate.This suggests the T to D mutation mimics amphetamine action.My studies have contributed significantly to our knowledge of the importance of juxtamembrane mutations to the conformation of DAT and NET, as well as knowledge of the responsiveness of transporter conformations to ligands and, crucially, to membrane gradients.This work demonstrates that the transporter, despite being a pump, can have heavily modified function at one gate without disruption of function at the other gate.