Melittin initiates dopamine transporter internalization and recycling in transfected HEK-293 cells.
The dopamine transporter removes the neurotransmitter from the synapse, regulating dopamine availability. The transporter can be internalized and its function is blocked by cocaine and other ligands. Melittin inhibits dopamine transporter function and causes internalization of the recombinant transporter in stably transfected HEK-293 cells, but the specific pathways for internalization and disposition of the transporter are unknown. Here we report that melittin treatment increased both transporter internalization and colocalization with clathrin, effects that were blocked by pretreatment with cocaine. Density gradient centrifugation revealed that melittin treatment caused the dopamine transporter to associate with a density fraction containing the early endosome marker Rab 5A. Confocal microscopy revealed that melittin treatment also increased transporter colocalization with Rab 5A and decreased colocalization with the late endosome marker Rab 7 and the recycling endosome marker Rab 11. Following 60 min of melittin treatment, the transporter was trafficked back to the membrane. By comparison, phorbol ester treatment increased transporter colocalization with early endosome antigen 1 and Rab 7 in a time-dependent manner. Cocaine treatment alone does not affect transporter trafficking in these cells. Results indicate multiple dopamine transporter internalization and recycling pathways that depend on transporter-ligand interactions and post-translational modifications.
Graduate Program in Neuroscience, VA Medical Center, Portland, OR 97239, USA.
Dove J
DJ
Keith
Katherine
K
Wolfrum
Amy J
AJ
Eshleman
Aaron
A
Janowsky
D019921:Biotinylation / Q000187:drug effects; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008555:Melitten / Q000378:metabolism / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D014162:Transfection
D050483:Dopamine Plasma Membrane Transport Proteins; D008555:Melitten; C016658:phorbolol myristate acetate; D013755:Tetradecanoylphorbol Acetate