title: Transmembrane topology and oligomeric structure of the high-affinity choline transporter.
abstract: The high-affinity choline transporter CHT1 mediates choline uptake essential for acetylcholine synthesis in cholinergic nerve terminals. CHT1 belongs to the Na(+)/glucose cotransporter family (SLC5), which is postulated to have a common 13-transmembrane domain core; however, no direct experimental evidence for CHT1 transmembrane topology has yet been reported. We examined the transmembrane topology of human CHT1 using cysteine-scanning analysis. Single cysteine residues were introduced into the putative extra- and intracellular loops and probed for external accessibility for labeling with a membrane-impermeable, sulfhydryl-specific biotinylating reagent in intact cells expressing these mutants. The results provide experimental evidence for a topological model of a 13-transmembrane domain protein with an extracellular amino terminus and an intracellular carboxyl terminus. We also constructed a three-dimensional homology model of CHT1 based on the crystal structure of the bacterial Na(+)/galactose cotransporter, which supports our conclusion of CHT1 transmembrane topology. Furthermore, we examined whether CHT1 exists as a monomer or oligomer. Chemical cross-linking induces the formation of a higher molecular weight form of CHT1 on the cell surface in HEK293 cells. Two different epitope-tagged CHT1 proteins expressed in the same cells can be co-immunoprecipitated. Moreover, co-expression of an inactive mutant I89A with the wild type induces a dominant-negative effect on the overall choline uptake activity. These results indicate that CHT1 forms a homo-oligomer on the cell surface in cultured cells.
authors.affiliation: Department of Pharmacology, Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo, Japan. t-okuda@umin.org
authors.forename: Takashi
authors.initials: T
authors.lastname: Okuda
authors.forename: Chieko
authors.initials: C
authors.lastname: Osawa
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Yamada
authors.forename: Kengo
authors.initials: K
authors.lastname: Hayashi
authors.forename: Shizue
authors.initials: S
authors.lastname: Nishikawa
authors.forename: Tomoko
authors.initials: T
authors.lastname: Ushio
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kubo
authors.forename: Motoyasu
authors.initials: M
authors.lastname: Satou
authors.forename: Haruo
authors.initials: H
authors.lastname: Ogawa
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Haga
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D002794:Choline / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D005799:Genes, Dominant; D057809:HEK293 Cells; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D020836:Protein Structure, Quaternary; D040681:Structural Homology, Protein; D027981:Symporters / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; C510731:SLC5A7 protein, human; D027981:Symporters; D003545:Cysteine; D002794:Choline