title: 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⁻) donor]; ONOO⁻ 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 dynamin-dependent 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.
authors.affiliation: Molecular Brain Research Group, Robarts Research Institute, London, Ontario N6A 5K8, Canada.
authors.forename: Leah K
authors.initials: LK
authors.lastname: Cuddy
authors.forename: Alexis C
authors.initials: AC
authors.lastname: Gordon
authors.forename: Stefanie A G
authors.initials: SA
authors.lastname: Black
authors.forename: Ewa
authors.initials: E
authors.lastname: Jaworski
authors.forename: Stephen S G
authors.initials: SS
authors.lastname: Ferguson
authors.forename: R Jane
authors.initials: RJ
authors.lastname: Rylett
mesh_terms: D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002794:Choline / Q000378:metabolism*; D002966:Clathrin / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D006426:Hemicholinium 3 / Q000493:pharmacokinetics; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008981:Molsidomine / Q000031:analogs & derivatives* / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D009447:Neuroblastoma / Q000473:pathology; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D030421:Peroxynitrous Acid / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D013997:Time Factors; D014162:Transfection; D014316:Tritium / Q000378:metabolism / Q000493:pharmacokinetics; D054875:Ubiquitination / Q000502:physiology; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002966:Clathrin; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D020030:Nitric Oxide Donors; C065637:choline transporter; D014316:Tritium; D030421:Peroxynitrous Acid; D006426:Hemicholinium 3; D009569:Nitric Oxide; C002385:linsidomine; D008981:Molsidomine; D020696:rab5 GTP-Binding Proteins; D002794:Choline; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde