Activity and subcellular trafficking of the sodium-coupled choline transporter CHT is regulated acutely by peroxynitrite

• Published in: Molecular pharmacology Vol. 73; no. 3; p. 801
• Main Authors: Pinthong, Metta, Black, Stefanie A G, Ribeiro, Fabiola M, Pholpramool, Chumpol, Ferguson, Stephen S G, Rylett, R Jane
• Format: Journal Article
• Language: English
• Published: United States 01-03-2008
• Subjects: Animals; Biotinylation; Cell Culture Techniques; Cell Line; Cell Line, Tumor; Cell Membrane - chemistry; Cell Membrane - metabolism; Choline - antagonists & inhibitors; Choline - metabolism; Cholinergic Agents - metabolism; Cholinergic Agents - pharmacology; Culture Media; Data Interpretation, Statistical; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; Gene Expression Regulation - drug effects; Gene Expression Regulation - physiology; Hemicholinium 3 - metabolism; Hemicholinium 3 - pharmacology; Humans; Inhibitory Concentration 50; Kidney - cytology; Kinetics; L-Lactate Dehydrogenase - analysis; Luminescence; Membrane Potentials - drug effects; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Molsidomine - analogs & derivatives; Molsidomine - pharmacology; Neuroblastoma - pathology; Nitrogen - metabolism; Oxidative Stress - drug effects; Peroxynitrous Acid - biosynthesis; Peroxynitrous Acid - metabolism; Protein Transport; Rats; Sodium - metabolism; Subcellular Fractions - metabolism; Time Factors; Transfection; Tyrosine - metabolism
• ISSN: 1521-0111
• DOI: 10.1124/mol.107.040881

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 IC(50) = 0.9 +/- 0.14 mM and t((1/2)) = 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.