Received for publication August 16, 2007.
Revised October 29, 2007.
Accepted for publication October 29, 2007.

ACTIVITY AND SUBCELLULAR TRAFFICKING OF THE SODIUM-COUPLED CHOLINE TRANSPORTER CHT IS REGULATED ACUTELY BY PEROXYNITRITE

Abstract

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 dose- and time-dependent manner with an IC₅₀ = 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-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.

Key words: Nitric oxide, Biogenic Amine, Fluorescence techniques, Oxidative stress/antioxidants, Excitotoxicity, neurodegeneration