Rab11 Supports Amphetamine-Stimulated Norepinephrine Transporter Trafficking

Abstract

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 AMPHinduced 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 (FIP2⌬C2) 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 (FIP2⌬C2) 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.

Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini

“…The z-scan also highlights the presence of NET in the plasma membrane of the neuronal somata. Notably, NET expression in the plasma membrane of the somata has not been reported in previous studies (16,40,41). Taken together, the results suggest that JHC 1-64 efficiently labels endogenous NET in cultured noradrenergic neurons with high signal/noise ratio.…”

“…by G q -coupled muscarinic acetylcholine receptors) can increase NET internalization and reduce NE transport capacity (11)(12)(13)(14). Furthermore, amphetamine has been shown to decrease NET surface expression, whereas cocaine was found to enhance NET availability (15)(16)(17). An elegant study by Galli and co-workers (16) found evidence that the amphetamine-induced decrease in NET surface levels is Rab11-dependent.…”

“…Furthermore, amphetamine has been shown to decrease NET surface expression, whereas cocaine was found to enhance NET availability (15)(16)(17). An elegant study by Galli and co-workers (16) found evidence that the amphetamine-induced decrease in NET surface levels is Rab11-dependent. The general NET trafficking properties and postendocytic sorting pattern, however, have not been investigated in detail.…”

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation

“…A marked constitutive internalization has also been shown for other members of the SLC6 transporter family such as the DAT, GLYT2, and norepinephrine transporter (11,12,42). However, the fate of constitutively internalized SLC6 transporters, and ultimately the consequences of the internalization, remains elusive.…”

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…These studies underscore the differential capacities of neuronal and nonneuronal hosts to support seemingly equivalent changes in transporter function through different mechanisms. In this regard, Matthies et al (2010) demonstrated reduced surface expression of NET in mouse brain slices and single varicosities of cultured SCG neurons after AMPH treatment. Future studies that extend this paradigm to an evaluation of the role of Ca 2+ /CaMKII mechanisms in basal and AMPH-modulated NET trafficking should yield important insights.…”