Rapid, transient effects of the protein kinase C activator phorbol 12-myristate 13-acetate on activity and trafficking of the rat high-affinity choline transporter.
Cholinergic neurons rely on the sodium-dependent choline transporter CHT to provide choline for synthesis of acetylcholine. CHT cycles between cell surface and subcellular organelles, but little is known about regulation of this trafficking. We hypothesized that activation of protein kinase C with phorbol ester modulates choline uptake by altering the rate of CHT internalization from or delivery to the plasma membrane. Using SH-SY5Y cells that stably express rat CHT, we found that exposure of cells to phorbol ester for 2 or 5 min significantly increased choline uptake, whereas longer treatment had no effect. Kinetic analysis revealed that 5 min phorbol ester treatment significantly enhanced V(max) of choline uptake, but had no effect on K(m) for solute binding. Cell-surface biotinylation assays showed that plasma membrane levels of CHT protein were enhanced following 5 min phorbol ester treatment; this was blocked by protein kinase C inhibitor bisindolylmaleimide-I. Moreover, CHT internalization was decreased and delivery of CHT to plasma membrane was increased by phorbol ester. Our results suggest that treatment of neural cells with the protein kinase C activator phorbol ester rapidly and transiently increases cell surface CHT levels and this corresponds with enhanced choline uptake activity which may play an important role in replenishing acetylcholine stores following its release by depolarization.
Black
S A G
SA
Robarts Research Institute, University of Western Ontario, London, Ontario, Canada.
Ribeiro
F M
FM
Ferguson
S S G
SS
Rylett
R J
RJ
D000109:Acetylcholine / Q000096:biosynthesis; D000818:Animals; D001921:Brain / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000187:drug effects* / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D050480:Plasma Membrane Neurotransmitter Transport Proteins / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000187:drug effects* / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D050480:Plasma Membrane Neurotransmitter Transport Proteins; C075746:Slc6a8 protein, rat; C016658:phorbolol myristate acetate; D011493:Protein Kinase C; D000109:Acetylcholine; D013755:Tetradecanoylphorbol Acetate