Neurotransmitter Transporters by Heinz Bönisch & Harald H. Sitte

Author:Heinz Bönisch & Harald H. Sitte
Language: eng
Format: epub
Publisher: Springer New York, New York, NY

Key words

Metabolic labelingPhosphoamino acid analysisphosphopeptide mappingPhosphospecific antibodies

1 Introduction

The SLC6 family of Na+/Cl−-dependent transporters includes carriers for dopamine (DA), serotonin (5-HT), norepinephrine (NE ), gamma-aminobutyric acid, taurine, creatine, as well as several amino acids [1]. The monoamines DA, 5-HT, and NE control many functions including movement, mood, motivation, cognition, and reward, and their respective transporters DAT, SERT, and NET are responsible for the spatial and temporal control of extraneuronal neurotransmitter levels by driving the reuptake of extracellular transmitter into presynaptic neurons . Disruption in brain monoamine levels has been associated with several diseases such as depression , bipolar disorder, anxiety , attention-deficit hyperactivity disorder, obsessive–compulsive disorder, Parkinson disease , and Tourette syndrome. In that light, monoamine transporters are the targets of several therapeutic drugs used to alter extraneuronal levels of these neurotransmitters in the management of these disorders. In addition, these transporters are the target of psychostimulant drugs including cocaine and amphetamines (amphetamine , methamphetamine, and 3,4,-methylenedioxy-N-methamphetamine) that elevate extraneuronal levels of monoamines .

Although monoamine neurotransmitter transporters were once thought to be static in terms of their activity and membrane levels, it is now clear that these transporters are regulated, providing neurons the ability to modify transport velocities in response to physiological stimuli. Transporter function is modulated through posttranslational modifications and interaction with binding partners that alter transporter kinetics, trafficking, and cell surface levels. A growing body of evidence suggests that disruption of these regulatory processes may be factors in related disease etiologies making the identification and characterization of these posttranslational modifications a high priority [2].

Our laboratory has examined the regulation of DAT by phosphorylation for several years [3–5] and more recently we have begun studying DAT palmitoylation and its effect on transporter activity, trafficking, and degradation [6]. We have employed strategic combinations of several methods to identify phosphorylation and palmitoylation sites in the transporter. These techniques include metabolic radiolabeling with [32P]H3PO4 or [3H]palmitic acid, phosphoamino acid analysis , 2D phosphopeptide mapping, in vitro N- and C-terminal peptide phosphorylation analysis, DAT site-specific mutagenesis , acyl-biotinyl exchange (ABE ) , and the generation of phosphospecific antibodies . The challenges in studying these DAT modifications lie in the fact that multiple phosphorylation and palmitoylation sites are present that are acted upon by multiple enzymes. These characteristics in addition to differences in expression levels of WT and mutant DAT proteins make it difficult to compare posttranslational modifications without very careful control of these variables.

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