22 research outputs found
Substrate-modulated unwinding of transmembrane helices in the NSS transporter LeuT:Recommended in F1000Prime as being of special significance in its field by F1000 Faculty Member Robert Vandenberg
The use of LeuT as a model in elucidating binding sites for substrates and inhibitors in neurotransmitter transporters
Defining proximity relationships in the tertiary structure of the dopamine transporter. Identification of a conserved glutamic acid as a third coordinate in the endogenous Zn(2+)-binding site.
Delineating structure-function relationships in the dopamine transporter from natural and engineered Zn2+ binding sites
Intracellular residues in the dopamine transporter critical for conformational isomerization, molecular gating and cocaine binding
A Conserved Salt Bridge between Transmembrane Segment 1 and 10 Constitutes an Extracellular Gate in the Dopamine Transporter
Neurotransmitter transporters play an important role in termination of synaptic transmission by mediating reuptake of neurotransmitter, but the molecular processes behind translocation are still unclear. The crystal structures of the bacterial homologue, LeuT, provided valuable insight into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the “thin gate” formed by an interaction between Arg-30 and Asp-404. In the human dopamine transporter (DAT), the corresponding residues are Arg-85 and Asp-476. Here, we present results supporting the existence of a similar interaction in DAT. The DAT R85D mutant has a complete loss of function, but the additional insertion of an arginine in opposite position (R85D/D476R), causing a charge reversal, results in a rescue of binding sites for the cocaine analogue [(3)H]CFT. Also, the coordination of Zn(2+) between introduced histidines (R85H/D476H) caused a ∼2.5-fold increase in [(3)H]CFT binding (B(max)). Importantly, Zn(2+) also inhibited [(3)H]dopamine transport in R85H/D476H, suggesting that a dynamic interaction is required for the transport process. Furthermore, cysteine-reactive chemistry shows that mutation of the gating residues causes a higher proportion of transporters to reside in the outward facing conformation. Finally, we show that charge reversal of the corresponding residues (R104E/E493R) in the serotonin transporter also rescues [(3)H](S)-citalopram binding, suggesting a conserved feature. Taken together, these data suggest that the extracellular thin gate is present in monoamine transporters and that a dynamic interaction is required for substrate transport