| Abstract No.: | B-B3031 |
| Country: | Canada |
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| Title: | INHIBITION OF HIGH-AFFINITY CHOLINE TRANSPORTER BY PEROXYNITRITE |
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| Authors/Affiliations: | 1 Alexis Gordon*; 1 Jane Rylett;
1 University of Western Ontario, London, ON, Canada
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| Content: | Cholinergic neurotransmission can be disrupted by oxidative stress, and this sensitivity may play a role in the pathology of Alzheimer disease (AD). In these cholinergic neurons, choline serves as a precursor for the synthesis of the neurotransmitter acetylcholine (ACh). As choline does not permeate the plasma membrane, ACh production is dependent on choline uptake mediated by the high-affinity, sodium-dependent choline transporter (CHT). This uptake is thought to be the rate-limiting step in ACh synthesis, and is sensitive to hemicholinium-3 (HC-3). As dysregulation of CHT can substantially reduce ACh synthesis and release, it is an important target for disruption in diseases such as AD in which cholinergic neurons are affected. One reactive species which may be responsible for such disruption is peroxynitrite (PN), a powerful oxidant implicated in the pathology of several neurodegenerative diseases, including AD. Objectives: Previous studies have shown a dose dependent inhibition of CHT by PN; the objective of this study was to identify a mechanism for this inhibition. Trafficking-independent effects of PN on CHT were determined using the L531A CHT mutant, which does not constitutively internalize. Materials and Methods: Human embryonic kidney cells (HEK) were engineered for stable expression of either CHT or L531A CHT, and treated with vehicle or the peroxynitrite donor 3-morpholinosydnonimine (SIN-1). Effect of PN on CHT activity was determined by incubating cells for 30 min with SIN-1 ranging from 0 to 1 mM SIN-1. Transporter activity was subsequently assayed by uptake of tritiated substrate. Time courses examining effect of SIN-1 on surface levels were obtained by incubation of cells with 1 mM SIN-1; cell surface transporter levels were then assayed by binding of tritiated hemicholinium-3 (HC-3), a specific CHT inhibitor. Results: Both wild type and L531A CHT show dose-dependent inhibition by SIN-1. Surface labelling with HC-3 shows a rapid decrease in cell surface levels of both wild type and L531A CHT, reaching 50% of control levels within 5 min. HC-3 binding kinetics show no change in KD of HC-3 binding, whereas there is a decrease in bmax. Conclusion: The proportional decrease in cell surface levels mirror the decrease in CHT activity, suggesting that PN effects are mediated in large part by CHT trafficking. The loss of cell surface L531A CHT was an unexpected and interesting result; further experiments will be performed to assess whether this loss is due to degradation or internalization along a non-clathrin-mediated pathway. |
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