RT Journal Article
SR Electronic
T1 A Juxtamembrane Mutation in the N Terminus of the Dopamine Transporter Induces Preference for an Inward-Facing Conformation
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 514
OP 524
DO 10.1124/mol.108.048744
VO 75
IS 3
A1 Bipasha Guptaroy
A1 Minjia Zhang
A1 Erica Bowton
A1 Francesca Binda
A1 Lei Shi
A1 Harel Weinstein
A1 Aurelio Galli
A1 Jonathan A. Javitch
A1 Richard R. Neubig
A1 Margaret E. Gnegy
YR 2009
UL http://molpharm.aspetjournals.org/content/75/3/514.abstract
AB 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 [3H]dopamine up-take but exhibited a higher basal dopamine efflux compared with hDAT or T62A-hDAT, as determined by measurements of [3H]dopamine efflux and amperometry. The high constitutive efflux in T62D-hDAT precluded the measurement of amphetamine-stimulated [3H]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 Zn2+ can rescue reduced DA uptake in mutant transporters that are predominantly inward-facing, micromolar concentrations of Zn2+ markedly potentiated [3H]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 [3H]dopamine uptake and [3H]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. The American Society for Pharmacology and Experimental Therapeutics