title: Regulation of the high-affinity choline transporter activity and trafficking by its association with cholesterol-rich lipid rafts.
abstract: The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.
authors.affiliation: Molecular Brain Research Group, Robarts Research Institute, Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada.
authors.forename: Leah K
authors.initials: LK
authors.lastname: Cuddy
authors.forename: Warren
authors.initials: W
authors.lastname: Winick-Ng
authors.forename: Rebecca Jane
authors.initials: RJ
authors.lastname: Rylett
mesh_terms: D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism*; D002789:Cholesterol Oxidase / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D003627:Data Interpretation, Statistical; D005260:Female; D005372:Filipin / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism; D006426:Hemicholinium 3 / Q000378:metabolism; D006801:Humans; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D051381:Rats; D013574:Synaptosomes / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D047392:beta-Cyclodextrins; C065637:choline transporter; C106354:flotillins; C108732:methyl-beta-cyclodextrin; D006426:Hemicholinium 3; D005677:G(M1) Ganglioside; D005372:Filipin; D002784:Cholesterol; D002789:Cholesterol Oxidase; D002794:Choline
keywords: cholesterol; high-affinity choline uptake; lipid rafts; trafficking; transport