| High-affinity choline uptake mediated by the Na+-dependent, hemicholinium (HC)-3-sensitive choline transporter CHT is rate-limiting in synthesis of the neurotransmitter acetylcholine in cholinergic neurons. CHT proteins traffic between the cell surface and subcellular organelles in both a constitutive and regulated manner, but regulation of CHT trafficking is not well-understood. Thus, our experiments focused on post-translational mechanisms regulating the subcellular distribution and activity of CHT. Our research examined regulation of CHT by protein kinase-C (PK-C) and identified protein binding partners for CHT.;Further investigation of PK-C-mediated CHT regulation used human embryonic kidney (HEK) 293 cells transiently-transfected to express FLAG-tagged wild-type CHT or CHT proteins having mutations at putative PK-C phosphorylation sites. In this experimental model, PMA decreased high-affinity choline uptake. Mutation of the serine-373 residue in CHT abolished PMA-mediated regulation of CHT activity and also reduced CHT protein levels, which correlated with reduced transcript levels. Both incorporation of [32P]-phosphate into CHT proteins and [3H]-hemicholinium-3 binding of transporters were unaltered by either PMA treatment or by mutation of potential phosphorylation sites examined in these studies.;Using a variety of methods, an interaction of the carboxyl-terminal tail of human CHT with the carboxyl-terminal region (CTR) of microtubule-associated protein 1A light chain 2 (LC2) was identified. LC2-CTR bound to several regions in the carboxyl-tail of CHT in vitro, with basic residues in this region of CHT playing an important role in binding of LC2-CTR. FLAG-CHT co-localized with myc-LC2-CTR in transfected neurons, and high-affinity choline uptake was modestly increased in HEK 293 cells co-expressing FLAG-CHT and myc-LC2-CTR.;Our studies help define the molecular regulation of CHT proteins. Information from studies such as these will aid in developing approaches to treat pathologies involving cholinergic deficiencies.;Our investigation of PK-C-mediated regulation of CHT trafficking using neural cells ectopically-expressing FLAG-tagged CHT revealed that the PK-C activator PMA acutely enhanced the choline uptake activity of CHT. Increased CHT activity correlated with increased cell surface CHT levels in response to PMA, which resulted from both decreased CHT endocytosis and enhanced delivery of transporters to the plasma membrane.;Keywords: CHT, high-affinity choline uptake, cholinergic, protein kinase-C, phosphorylation, protein trafficking, subcellular distribution, cell surface biotinylation, hemicholinium-3, protein-protein interaction, yeast two-hybrid, co-immunoprecipitation, scanning peptide array, microtubule-associated protein 1A, light chain 2. |
