Review
Biogenic amine transporters: regulation in flux

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Abstract

Following vesicular release, the biogenic amine neurotransmitters dopamine, norepinephrine and serotonin are actively cleared from extracellular spaces by presynaptic transporters. These transporters interact with multiple psychoactive agents including cocaine, amphetamines and antidepressants. Recent findings indicate that amine reuptake is likely to be a tightly regulated component of synaptic plasticity rather than a constitutive determinant of transmitter clearance. Protein kinase C activation and transporter phosphorylation have been linked to regulatory protein trafficking, and both phosphorylation and trafficking may be influenced by transporter ligands. Recognition that transmitters, antagonists and second messengers can modify the intrinsic activity, surface expression or protein levels of amine transporters raises new questions about the fundamental nature of drug actions in vivo. The theory that dysregulation of transporters may contribute to disease states is supported by the recent discovery that a coding mutation in the human norepinephrine transporter contributes to orthostatic intolerance.

Introduction

The biogenic amine neurotransmitters dopamine (DA), norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) are very simple molecules with highly complex actions in the peripheral and central nervous systems ranging from the control of heart rate to the coloring of mood. Pharmacologists have been fascinated by the amines for decades, as the management of amine production, action or inactivation figures prominently in the treatment of autonomic, emotional and cognitive disturbances. The past decade began with an elucidation of the genes responsible for clearance of amines from the synaptic cleft [1]. Evaluation of the amino acid sequence of cloned NE transporters (NETs) [2] led to the prediction that transporters comprised 12 transmembrane domains (TMDs), in register with the hydrophobic sequences of the GAT1 GABA transporter [3]. This model, subsequently adopted for cloned DA transporters (DATs) and 5-HT transporters (SERTs), identified a large hydrophilic extracellular loop (EL2) between TMD3 and 4 and placed the NH2 and COOH termini intracellularly. The initial cloning of amine transporters also revealed multiple protein phosphorylation sites on the cytoplasmic domains. With a growing awareness of the sensitivity of amine transport to kinase activation and the generation of heterologous expression systems as well as transporter-specific antibodies suitable for biochemical and cell biologic studies, amine transporter biology has entered a new era of discovery. Recent studies, which will be reviewed here, indicate that regulation of transporter function and surface expression can be rapidly modulated and that drugs previously studied only for their impact on transport activity may have unrecognized capacities for influencing transporter regulation. The impact that such regulatory processes have on human physiology and behavior may be revealed by recent studies identifying the first coding mutation in an amine transporter that supports a disease process.

Section snippets

Regulating amine reuptake: general thoughts

Pharmacological studies with amine transporter knockout mice reveal a requirement for presynaptic amine transporter expression for normal transmitter clearance, presynaptic transmitter homeostasis and drug responses 4, 5••, 6••. These studies also indicate that cells must make both quantitative (how much, how active) and qualitative (which transporter to express, where to locate them) decisions to achieve appropriate transporter contributions for the particular neurotransmitter signals they

Transporter trafficking: skimming the surface of transporter regulation

Prior to the 1990s, methods for investigating the distribution and surface expression of biogenic amine transporters were unavailable, perhaps perpetuating the concept that surface expression is set at a constitutive level and is not subject to modulation. In the mid 1990s, with the arrival of transporter-specific antibodies and the development of heterologous expression models, researchers began to report evidence for changes in transport capacity that ensue rapidly (1–30 min) following the

Transporter surface expression

Recent studies with transporter-specific antibodies and/or epitope-tagged constructs have yielded evidence that changes in transport capacity arise from changes in transporter surface distribution. Initial evidence for altered trafficking as a route for amine transporter modulation has come from studies of SERT proteins in stably transfected HEK-293 cells [8] and homologous GAT1 GABA transporters expressed in Xenopus oocytes 9, 10. Apparsundaram and coworkers have visualized a PKC-dependent

Transporter endocytosis: recycling or degradation?

What is the fate of amine transporters once they leave the plasma membrane? Melikian and Buckley [13••], using transfected PC-12 cells, report a colocalization of internalized DAT proteins with transferrin receptors, proteins known to actively recycle to the plasma membrane. These findings suggest the passage of DATs through a recycling endosomal compartment. In contrast, Daniels and Amara [15••] report the movement of internalized DATs to lysosomes for degradation. It is possible that

Regulation of endogenous transporters

The studies reviewed above use heterologous expression in transfected cell lines or oocytes assayed in vitro. Although more limited in number, studies with isolated tissue preparations reveal similar acute transporter regulatory events that not only can be triggered by activated protein kinases but also by direct auto- and heteroreceptor activation. Apparsundaram and coworkers report muscarinic M3ACh-receptor-triggered internalization of NET proteins in SK-N-SH cells, partially mediated by a

Transporter phosphorylation: the signal for transporter internalization?

Many signaling proteins, including receptors and ion channels, are modulated via direct protein phosphorylation. Over the past several years, evidence has accumulated to support a role of biogenic amine transporter phosphorylation in transporter regulation. Huff, Vaughan and coworkers first demonstrated that DATs expressed in LLC-PK1 cells [24] and in synaptosomes [25] can be phosphorylated following PKC activation and protein phosphatase inhibition. Recently, Ramamoorthy and coworkers

Substrate-modulated transporter phosphorylation

It is possible that the key kinase target responsible for transporter redistribution is not the transporter per se but an associated protein or protein complex. Recent studies of substrate modulation of SERT phosphorylation and trafficking by Ramamoorthy and Blakely [27••], strengthen the case for direct transporter phosphorylation as the key to transporter trafficking. These authors report that 5-HT and amphetamine substrates block the ability of SERTs to become phosphorylated following PKC

Transporter-associated proteins

Phosphorylation-dependent desensitization and trafficking of G-protein-coupled receptors involves dynamic physical associations of receptors with kinases, phosphatases and adaptor proteins. We and others have proposed analogous schemes for the regulated trafficking of biogenic amine transporters 7, 29. Recent findings from our group (A Bauman, unpublished data) reveal associations between SERT, NET and DAT proteins and the catalytic subunit (c) of PP2A (PP2Ac). SERT–PP2Ac associations can be

Transporters as homomultimeric complexes

The models discussed above relate to heteromeric assemblies of transporters with other membrane or cytosolic proteins. Recent evidence indicates, however, that homomultimeric assemblies of transporter proteins may be at the core of the transporter complex, and such assemblies must be considered as we attempt to visualise the coassembly of transporter regulators. Chang and coworkers [31] exploited heterologously expressed, concatamer cDNA constructs to lend functional support to the idea of

Trafficking-independent regulation of biogenic amine transporters

Cells could silence or activate surface transporters, rather than shuttling active carriers from one compartment to another, to achieve an equivalent functional result. Ion channels and G-protein-coupled receptors are subject to trafficking-independent modes of regulation, and it would be surprising if transporters did not also avail themselves of this opportunity. Detection of such regulation requires that one be able to demonstrate either altered function without a change in surface

Transporter polarity: putting carriers in their place

Microscopic examination of DAT, NET and SERT localization in native tissues reveals targeting to axonal membranes and selected dendritic compartments but not to cell soma membranes 40, 41•, 42•. This subcellular localization or polarized membrane expression presumably indicates a need to place transporters proximal to release sites. For NETs, Schroeter and coworkers report a punctate localization along many noradrenergic fibers [42], though other fibers exhibit more continuous

Transporter genes as candidates for autonomic, emotional and cognitive disorders

The recent progress in the cloning of human transporter genes and the observed changes in amine levels, receptors, behavior and drug responses of transporter-deficient mice have given researchers new impetus to look for genetic variants in transporter proteins that underlie behavioral or autonomic disorders. Promoter variants have been uncovered in the human SERT gene [45] that have an impact on mRNA and protein expression in vitro and in vivo. These variants have been linked to anxiety traits

Conclusions

The past few years have been an active time for researchers evaluating the structure, function and regulation of transporter genes and proteins. Though much work remains to be done, we no longer consider transport as a constitutive property of synaptic membranes but rather as an actively regulated element of aminergic signaling. Transporters appear to be susceptible to regulatory influences mediated by protein associations and activated signal transduction cascades, and we are beginning to

Update

Following the completion of this review, Xu and coworkers [56••] have reported the first findings with NET knockout mice. These mice, as expected, have a diminished clearance capacity for NE and exhibit behaviors seen with antidepressant-treated mice. Moreover, compensatory changes in dopaminergic signaling are evident including locomotor hyperresponsiveness to psychostimulants (cocaine and amphetamine) and DA receptor supersensitivity.

Acknowledgements

The authors acknowledge the support of National Institutes of Health awards DA 07390 and MH 58291 (RD Blakely).

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • 1.

    • of special interest

  • 2.

    •• of outstanding interest

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