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Mutation of the Threonine (Thr) in the RETW juxtramembrane sequence in DAT and NET differentially alters transporter conformation, agonist and antagonist sensitivity
Author(s) -
Fraser Rheaclare,
Guptaroy Bipasha,
Desai Aalisha,
Gnegy Margaret E.
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.1083.6
Subject(s) - norepinephrine transporter , mutant , chemistry , allosteric regulation , dopamine transporter , transporter , mutation , efflux , antagonist , biochemistry , microbiology and biotechnology , enzyme , biology , gene , receptor
We investigated the role of the Thr residue within the highly conserved RETW sequence in the N‐terminal region of the dopamine (DA) and norepinephrine transporters (DAT and NET). Mutation of this Thr to Ala (A) or Asp (D) altered DAT and NET surface expression and [ 3 H] DA uptake. D mutations increased basal and abrogated amphetamine (AMPH)‐stimulated DA efflux. We posit that the D mutation favors an inward facing conformation, which readily releases substrate; while wildtype and A mutants are more outward facing. Substrate affinity for competition of [ 3 H] DA uptake and inhibitor binding is robustly increased in DAT and NET D mutant cells. Substrate binding shows temperature dependence. However, potency of the inhibitors cocaine (COC) and benztropine (BZT) to block uptake is reduced in the D mutant of DAT, but not NET. COC and BZT had differential affects on displacement of inhibitor binding in the DAT D mutant but not in the NET mutants. Our work suggests this Thr residue is important for differentially regulating function of substrates and inhibitors and profoundly affects the configuration of DAT and NET. It may be a potential target for AMPH action. This work was supported by DA011679 & 5T32DA007281.