The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles.

Ribeiro, F M; Alves-Silva, J; Volknandt, W; Martins-Silva, C; Mahmud, H; Wilhelm, A; Gomez, M V; Rylett, R J; Ferguson, S S G; Prado, V F; Prado, M A M

Ribeiro, F M; Alves-Silva, J; Volknandt, W; Martins-Silva, C; Mahmud, H; Wilhelm, A; Gomez, M V; Rylett, R J; Ferguson, S S G; Prado, V F; Prado, M A M.

Afiliação

Ribeiro FM; Laboratório de Neurobiologia Molecular, Departamento de Bioquímica-Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

J Neurochem ; 87(1): 136-46, 2003 Oct.

Article em En | MEDLINE | ID: mdl-12969261

RESUMO

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.

Assuntos

Clatrina/metabolismo; Endocitose/fisiologia; Endossomos/metabolismo; Hemicolínio 3/farmacologia; Proteínas de Membrana Transportadoras/metabolismo; Vesículas Sinápticas/metabolismo; Proteínas de Transporte Vesicular; Animais; Proteínas de Transporte/genética; Proteínas de Transporte/metabolismo; Linhagem Celular; Humanos; Rim/citologia; Rim/metabolismo; Proteínas de Membrana/genética; Proteínas de Membrana/metabolismo; Proteínas de Membrana Transportadoras/efeitos dos fármacos; Proteínas de Membrana Transportadoras/genética; Camundongos; Neurônios/citologia; Neurônios/metabolismo; Proteínas R-SNARE; Proteínas Recombinantes de Fusão/genética; Proteínas Recombinantes de Fusão/metabolismo; Sinaptossomos/metabolismo; Proteínas Vesiculares de Transporte de Acetilcolina

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Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Proteínas de Membrana Transportadoras / Endossomos / Vesículas Sinápticas / Clatrina / Proteínas de Transporte Vesicular / Endocitose / Hemicolínio 3 Tipo de estudo: Diagnostic_studies Limite: Animals / Humans Idioma: En Revista: J Neurochem Ano de publicação: 2003 Tipo de documento: Article País de afiliação: Brasil País de publicação: Reino Unido

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