Conserved serine residues in serotonin transporter contribute to high-affinity cocaine binding.
Serotonin transporter (SERT) is one of the key protein targets of cocaine. Despite intensive studies, it is not clear where cocaine binds to its targets and what residues are involved in cocaine binding. We have cloned the serotonin transporter from silkworm (Bombyx mori, bmSERT). When expressed in cultured cells, bmSERT is over 20-fold less sensitive to cocaine than Drosophila melanogaster SERT (dmSERT). We performed species-scanning mutagenesis using bmSERT and dmSERT. There are two adjacent threonine residues in transmembrane domain 12 of bmSERT where the corresponding residues are two serines in dmSERT and in all known mammalian monoamine transporters. Replacing the serine residues with threonines in dmSERT reduces cocaine sensitivity; while switching the two threonine residues in bmSERT to serines increased cocaine sensitivity. Mutations at the corresponding residues in dopamine transporter also changed cocaine affinity. Our results suggest that the conserved serine residues in SERT contribute to high-affinity cocaine binding.
Gu
Howard H
HH
Department of Pharmacology, The Ohio State University College of Medicine, 333 West 10th Avenue, Columbus, OH 43210, USA. gu.37@osu.edu
Wu
Xiaohong
X
Han
Dawn D
DD
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000378:metabolism*; D003042:Cocaine / Q000378:metabolism*; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D029721:Drosophila Proteins; D011993:Recombinant Fusion Proteins; C493233:SerT protein, Drosophila; D050486:Serotonin Plasma Membrane Transport Proteins; D013912:Threonine; D012694:Serine; D003042:Cocaine