Evidence for Distinct Sodium-, Dopamine-, and Cocaine-dependent Conformational Changes in Transmembrane Segments 7 and 8 of the Dopamine Transporter

Abstract

؉ . Because dopamine binding is believed to be Na ؉ -independent, this suggests that dopamine induces a transport-associated conformational change that decreases the reactivity of M371C with MTSET. In contrast to M371C, cocaine decreased the reaction rate of A399C with MTSET, whereas dopamine had no effect. The protection by cocaine can either reflect that Ala-399 lines the cocaine binding crevice or that cocaine induces a conformational change that decreases the reactivity of A399C. The present findings add new functionality to the TM7/8 region by providing evidence for the occurrence of distinct Na ؉ -, substrate-, and perhaps inhibitor-induced conformational changes critical for the proper function of the transporter.

Homozygous loss-of-function mutations in the gene encoding the dopamine transporter are associated with infantile parkinsonism-dystonia

“…Of the 2 helical portions of extracellular loop 4, EL4b, the position of the second missense mutation, Pro395, is in the helical portion closer to transmembrane 8. Pro395 lies just 1 helix turn above Ala399, a residue that, like Met371, points outward from the entire transporter structure and thus is easily accessible to solvent bathing the outside of the protein (14). The interaction between a sulfhydryl reagent from the outside and position 399 was not blocked in the presence of dopamine, whereas the interaction at the 371 position was highly sensitive to dopamine, which caused conformational changes, burying the residue away from solvent exposure.…”

“…Among the positions of the 2 IPD-associated missense mutations reported here, Leu368 in hDAT was in transmembrane domain 7 toward the exterior, many helix turns above the residue (Asn353) that presumably interacts with sodium ( Figure 8). Leu368 is 1 helix turn under Met371 and has been shown to be conformationally sensitive to dopamine transport (14). Of the 2 helical portions of extracellular loop 4, EL4b, the position of the second missense mutation, Pro395, is in the helical portion closer to transmembrane 8.…”

Infantile parkinsonism-dystonia: a dopamine “transportopathy”

“…Indeed, in heterologous cell studies, Kurian et al (14) show that both DAT mutations resulted in markedly impaired dopamine uptake, supporting a loss-of-function mechanism underlying disease pathogenesis, and one mutation also decreased the binding affinity of dopamine to DAT ( Figure 1B). The region of DAT harboring these mutations has been shown to be sensitive conformationally to a number of different agents, including Na + , cocaine, and dopamine (12). Since the mutations occur at residues highly conserved among other SLC6 transporters, a more general functional role is also possible.…”

“…Though the SLC6 protein family is very large and extensive, both phylogenetically and within species (5), the DAT has been among the most intensively studied members because of its functional relationships to disease, therapeutic agents, and drugs of abuse (8)(9)(10)(11)(12)(13). In this issue of the JCI, Kurian et al (14) have provided the first report of loss-of-function mutations in the SLC6A member 3 (SLC6A3; also known as DAT1) gene that directly links DAT dysfunction to a form of human autosomal recessive infantile parkinsonism-dystonia.…”

LeuT: A prokaryotic stepping stone on the way to a eukaryotic neurotransmitter transporter structure

“…Critical residues residing within the extracellular-facing half of the protein include an arginine in TM1 of GAT1, 129 and an aspartate or glutamate in TM10 of SERT. 130 Also essential are sections of EL2 in SERT/NET 66 and DAT 53 and parts of EL4 in GAT1, 131 DAT, 68,132 GlyT1b 69 and SERT. 133 Critical residues residing within the intracellular-facing half of the protein include an arginine and tryptophan in the N-terminal domain/TM1 of GAT1, 134 an asparate in TM8 of DAT, 135 and a tyrosine in the intracellular loop between TMs 6 and 7 of DAT.…”