| Abstract No.: | B-B2027 |
| Country: | Canada |
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| Title: | ACUTE TREATMENT WITH THE PHORBOL ESTER PMA INCREASES THE ACTIVITY AND PLASMA MEMBRANE LOCALIZATION OF THE SODIUM-DEPENDENT CHOLINE TRANSPORTER CHT |
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| Authors/Affiliations: | 1 Stefanie Black*; 1 R Jane Rylett;
1 University of Western Ontario, London, ON, Canada
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| Content: | Background: Choline uptake into cholinergic neurons by the sodium-dependent, hemicholinium-3 (HC-3)-sensitive choline transporter (CHT) is rate-limiting in acetylcholine synthesis. CHT cycles between the cell surface and intracellular vesicles, suggesting that trafficking of CHT determines the number of transporters at the surface for uptake of choline. Treatments that alter phosphorylation, such as blockade of protein phosphatases or activation of protein kinases, change the number of CHT proteins at the surface, although the mechanism(s) involved remain undefined.
Objectives: We aim to characterize the mechanism by which phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, alters choline uptake. We hypothesize that PMA modulates choline uptake by changing the rates of internalization and/or recycling of CHT.
Materials and Methods: For our studies, we are using SH-SY5Y cells stably expressing FLAG-tagged rat CHT. Time course and dose response experiments were performed to investigate the effect of PMA on choline uptake. The effect of PMA treatment on the maximal velocity of choline uptake (Vmax) and affinity for choline (Km) was assessed in choline uptake kinetic experiments with choline concentrations of 0.1-5 uM. Cell-surface biotinylation was used to investigate changes in plasma membrane levels of CHT protein following PMA treatment. Treatment of cells with 0.1% v/v dimethyl sulfoxide (DMSO) was used as a control in all experiments.
Results: Treatment with 100 nM or 1 uM PMA for 2 or 5 min increased choline uptake. In kinetic experiments, following 5 min treatment with 100 nM PMA, Vmax was increased, but Km remained unchanged compared to DMSO controls. Cell-surface biotinylation showed that incubation with 100 nM PMA for 5 min increased surface levels of CHT; however, 2 min treatment with 100 nM did significantly change CHT surface levels.
Conclusion: Our results suggest that PMA enhances choline uptake by increasing the amount of CHT at the plasma membrane. Alterations in surface levels of CHT could result from changes in internalization and/or recycling of CHT. Cell surface biotinylation followed by incubation of cells to allow internalization or recycling will be used to determine if PMA treatment alters either or both of these processes for CHT.
This work is supported by CIHR grant to RJR and CIHR doctoral research award to SAGB. |
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