title: A juxtamembrane mutation in the N terminus of the dopamine transporter induces preference for an inward-facing conformation.
abstract: The human dopamine transporter (hDAT) regulates synaptic dopamine (DA) levels and is the site of action of abused and therapeutic drugs. Here we study the effect of a threonine residue (Thr62 in hDAT) that is highly conserved within a canonical phosphorylation site (RETW) in the juxtamembrane N-terminal region of monoamine transporters. In stably transfected human embryonic kidney 293T cells, expression of T62D-hDAT was reduced compared with hDAT or T62A-hDAT. T62D-hDAT displayed dramatically reduced [(3)H]dopamine up-take but exhibited a higher basal dopamine efflux compared with hDAT or T62A-hDAT, as determined by measurements of [(3)H]dopamine efflux and amperometry. The high constitutive efflux in T62D-hDAT precluded the measurement of amphetamine-stimulated [(3)H]dopamine efflux, but when dopamine was added internally into voltage-clamped T62D-hDAT cells, amphetamine-induced efflux comparable with hDAT was detected by amperometry. In accordance with findings that Zn(2+) can rescue reduced DA uptake in mutant transporters that are predominantly inward-facing, micromolar concentrations of Zn(2+) markedly potentiated [(3)H]dopamine uptake in T62D-hDAT and permitted the measurement of amphetamine-stimulated dopamine efflux. These results suggest that T62D-hDAT prefers an inward-facing conformation in the transition between inward- and outward-facing conformations. For T62A-hDAT, however, the measured 50% reduction in both [(3)H]dopamine uptake and [(3)H]dopamine efflux was consistent with a slowed transition between inward- and outward-facing conformations. The mechanism underlying the important functional role of Thr62 in hDAT activity suggested by these findings is examined in a structural context using dynamic simulations of a three-dimensional molecular model of DAT.
authors.affiliation: Department of Pharmacology, 2220E MSRBIII, University of Michigan Medical School, Ann Arbor, MI 48109-0632, USA.
authors.forename: Bipasha
authors.initials: B
authors.lastname: Guptaroy
authors.forename: Minjia
authors.initials: M
authors.lastname: Zhang
authors.forename: Erica
authors.initials: E
authors.lastname: Bowton
authors.forename: Francesca
authors.initials: F
authors.lastname: Binda
authors.forename: Lei
authors.initials: L
authors.lastname: Shi
authors.forename: Harel
authors.initials: H
authors.lastname: Weinstein
authors.forename: Aurelio
authors.initials: A
authors.lastname: Galli
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Javitch
authors.forename: Richard R
authors.initials: RR
authors.lastname: Neubig
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Gnegy
mesh_terms: D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D019943:Amino Acid Substitution; D000661:Amphetamine / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003198:Computer Simulation; D017124:Conserved Sequence / Q000235:genetics; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D013816:Thermodynamics; D013912:Threonine / Q000235:genetics
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D013912:Threonine; D000661:Amphetamine; D000409:Alanine; D004298:Dopamine