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Activity and Subcellular Trafficking of the Sodium-Coupled Choline Transporter CHT Is Regulated Acutely by Peroxynitrite

Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada (M.P., S.A.G.B., S.S.G.F., R.J.R.); Cell Biology Research Group, Robarts Research Institute, London, Ontario, Canada (M.P., S.A.G.B., F.M.R., S.S.G.F., R.J.R.); and Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand (M.P., C.P.)

Excess formation of nitric oxide and superoxide by-products (peroxynitrite, reactive oxygen, and reactive nitrogen species) attenuates cholinergic transmission potentially having a role in Alzheimer disease pathogenesis. In this study, we investigated mechanisms by which acute exposure to peroxynitrite impairs function of the sodium-dependent hemicholinium-3 (HC-3)-sensitive choline transporter (CHT) that provides substrate for acetylcholine synthesis. The peroxynitrite generator 3-morpholinosydnonimine (SIN-1) acutely inhibited choline uptake in cells stably expressing FLAG-tagged rat CHT in a dose- and time-dependent manner, with an IC₅₀ = 0.9 ± 0.14 mM and t = 4 min. SIN-1 significantly reduced V_max of choline uptake without altering the K_m. This correlated with a SIN-1-induced decrease in cell surface CHT protein, observed as lowered levels of HC-3 binding and biotinylated CHT at the plasma membrane. It is noteworthy that short-term exposure of cells to SIN-1 accelerated the rate of internalization of CHT from the plasma membrane, but it did not alter return of CHT back to the cell surface. SIN-1 did not disrupt cell membrane integrity or cause cell death. Thus, the inhibitory effect of SIN-1 on choline uptake activity and HC-3 binding was related to enhanced internalization of CHT proteins from the plasma membrane to subcellular organelles.

Address correspondence to: Dr. Rebecca Jane Rylett, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada N6A 5C1. E-mail: jane.rylett{at}schulich.uwo.ca