Rab11 supports amphetamine-stimulated norepinephrine transporter trafficking.
The norepinephrine transporter (NET) is a presynaptic plasma membrane protein that mediates reuptake of synaptically released norepinephrine. NET is also a major target for medications used for the treatment of depression, attention deficit/hyperactivity disorder, narcolepsy, and obesity. NET is regulated by numerous mechanisms, including catalytic activation and membrane trafficking. Amphetamine (AMPH), a psychostimulant and NET substrate, has also been shown to induce NET trafficking. However, neither the molecular basis nor the nature of the relevant membrane compartments of AMPH-modulated NET trafficking has been defined. Indeed, direct visualization of drug-modulated NET trafficking in neurons has yet to be demonstrated. In this study, we used a recently developed NET antibody and the presence of large presynaptic boutons in sympathetic neurons to examine basal and AMPH-modulated NET trafficking. Specifically, we establish a role for Rab11 in AMPH-induced NET trafficking. First, we found that, in cortical slices, AMPH induces a reduction in surface NET. Next, we observed AMPH-induced accumulation and colocalization of NET with Rab11a and Rab4 in presynaptic boutons of cultured neurons. Using tagged proteins, we demonstrated that NET and a truncated Rab11 effector (FIP2DeltaC2) do not redistribute in synchrony, whereas NET and wild-type Rab11a do. Analysis of various Rab11a/b mutants further demonstrates that Rab11 regulates NET trafficking. Expression of the truncated Rab11a effector (FIP2DeltaC2) attenuates endogenous Rab11 function and prevented AMPH-induced NET internalization as does GDP-locked Rab4 S22N. Our data demonstrate that AMPH leads to an increase of NET in endosomes of single boutons and varicosities in a Rab11-dependent manner.
Matthies
Heinrich J G
HJ
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. heinrich.j.matthies@vanderbilt.edu
Moore
Jessica L
JL
Saunders
Christine
C
Matthies
Dawn Signor
DS
Lapierre
Lynne A
LA
Goldenring
James R
JR
Blakely
Randy D
RD
Galli
Aurelio
A
D018663:Adrenergic Agents / Q000494:pharmacology*; D000661:Amphetamine / Q000494:pharmacology*; D000818:Animals; D019921:Biotinylation; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D005728:Ganglia, Sympathetic / Q000166:cytology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009154:Mutation; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D015398:Signal Transduction; D013572:Synaptic Vesicles / Q000187:drug effects* / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D020695:rab4 GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D018663:Adrenergic Agents; D000661:Amphetamine; D002352:Carrier Proteins; D008565:Membrane Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D020691:rab GTP-Binding Proteins; D020695:rab4 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C543424:Rab11fip2 protein, rat