Mol #40881 1 Activity and Subcellular Trafficking of the Sodium-coupled Choline Transporter Cht Is Regulated Acutely by Peroxynitrite

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 SIN-1 acutely inhibited choline uptake in cells stably-expressing FLAG-tagged rat CHT in a doseand time-dependent manner with an IC50 = 0.9 ± 0.14 mM and t1/2 = 4 min. SIN-1 significantly reduced Vmax of choline uptake without altering the Km. This correlated with a SIN-1-induced decrease in cell surface CHT protein, observed as lowered levels of HC-binding and biotinylated-CHT at the plasma membrane. Importantly, acute exposure of cells to SIN-1 accelerated the rate of internalization of CHT from the plasma membrane, but 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. This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on October 30, 2007 as DOI: 10.1124/mol.107.040881 at A PE T Jornals on Jauary 3, 2018 m oharm .aspeurnals.org D ow nladed from