Peroxynitrite Donor SIN-1 Alters High-Affinity Choline Transporter Activity by Modifying Its Intracellular Trafficking

Abstract

Sodium-coupled, high-affinity choline transporters (CHTs) are inhibited by 3-morpholinosydnonimine (SIN-1) [peroxynitrite (ONOO Ϫ ) donor]; ONOOϪ can be produced from nitric oxide and reactive oxygen species during neurodegeneration. SIN-1 rapidly increases CHT internalization from the cell surface, and this correlates with decreased choline uptake. This study addresses mechanisms by which SIN-1 inhibits CHT function in human neuronal SH-SY5Y cells. Thus, mutant L531A-CHT, which does not constitutively internalize into cells by a clathrin-mediated process, is resistant to SIN-1 effects. This suggests that CHT inhibition is not due to oxidative-nitrosative inactivation of the protein and that decreased levels of cell surface CHT in SIN-1-treated cells is related to alterations in its trafficking and subcellular disposition. Dominant-negative proteins AP180C and dynamin-K44A, which interfere with clathrin-mediated and dynamindependent endocytosis, respectively, attenuate CHT inhibition by SIN-1. CHT in both vehicle-and SIN-1-treated cells colocalizes with Rab7, Rab9, and Lamp-1 in late endosomes and lysosomes to a similar extent. Lysosome inhibitors increase choline uptake, suggesting that CHT proteins are normally degraded by lysosomes, and this is not altered by oxidative stress. Unexpectedly, inhibitors of proteasomes, but not lysosomes, attenuate SIN-1-mediated inhibition of choline uptake, indicating that proteasomal degradation plays a role in regulating CHT disposition in SIN-1-treated cells. SIN-1 treatment also enhances CHT ubiquitination. Thus, CHT inhibition in SIN-1-treated cells is mediated by proteasomal degradation, which differs from inhibitory mechanisms for some neurotransmitter transporters under similar conditions. Increased oxidative-nitrosative stress in the microenvironment of cholinergic nerve terminals would diminish cholinergic transmission by reducing choline availability for ACh synthesis.

Choline transporter‐like protein 4 (CTL4) links to non‐neuronal acetylcholine synthesis

“…; Cuddy et al . ). Whether similar localization of CTL4 in analogous compartments (e.g., secretory vesicles, endosomes, or cell membrane) plays a role in non‐neuronal ACh synthesis remains to be determined.…”

Toll-Like Receptor 4–Mediated Nuclear Factor Kappa B Activation Is Essential for Sensing Exogenous Oxidants to Propagate and Maintain Oxidative/Nitrosative Cellular Stress

“…The presence of multiple unidentified protein bands in the immuno-blot confirmed the efficacy of PN-mediated protein nitration at the tyrosine residues. Moreover, PN (1 mM) was more effective in producing nitrated proteins than equimolar concentration SIN-1, an agent that generates and donates PN in situ [49].…”

Unresponsive Choline Transporter as a Trait Neuromarker and a Causal Mediator of Bottom-Up Attentional Biases

“…After stimulation in STs, intracellular CHTs may be modified (e.g., phosphorylation, ubiquitylation, or nitrosylation) so that they are rendered unavailable for synaptic plasma membrane relocation (Gates et al, 2004;Ribeiro et al, 2006;Cuddy et al, 2012;Yamada et al, 2012;Hartnett et al, 2014). As total synaptosomal CHT densities did not differ between the phenotypes, we assume that subcellular CHT distribution anomalies observed in STs, and the associated failure of CHTs to increase choline transport in response to stimulation, reflect an alteration in distribution or post-translational modifications, rather than degradation.…”