Elsevier

Neuroscience

Volume 170, Issue 2, 13 October 2010, Pages 408-416
Neuroscience

Cellular and Molecular Neuroscience
Research Paper
Syntaxin 1A regulates dopamine transporter activity, phosphorylation and surface expression

https://doi.org/10.1016/j.neuroscience.2010.07.025Get rights and content

Abstract

We investigated the functional relationship between the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein syntaxin 1A (syn 1A) and the dopamine transporter (DAT) by treating rat striatal tissue with Botulinum Neurotoxin C (BoNT/C) and co-transfecting syn 1A with DAT in non-neuronal cells, followed by analysis of DAT activity, phosphorylation, and regulation. Treatment of striatal slices with BoNT/C resulted in elevated dopamine (DA) transport Vmax and reduced DAT phosphorylation, while heterologous co-expression of syn 1A led to reduction in DAT surface expression and transport Vmax. Syn 1A was present in DAT immunoprecipitation complexes, supporting a direct or indirect interaction between the proteins. Phorbol ester regulation of DA transport activity was retained in BoNT/C-treated synaptosomes and syn 1A transfected cells, demonstrating that protein kinase C (PKC) and syn 1A effects occur through independent processes. These findings reveal a novel mechanism for regulation of DAT activity and phosphorylation, and suggest the potential for syn 1A to impact DA neurotransmission through effects on reuptake.

Section snippets

Materials

Carrier free 32PO4 was from MP Biomedicals, Solon, OH, USA; [3H]DA (41 Ci/mmol) and high range Rainbow molecular mass standards were from GE Healthcare BioSciences Corp., Piscataway, NJ, USA; Botulinum Neurotoxin C was purchased from Metabiologicals Inc., Madison, WI, USA; PMA was from EMD Chemicals, Gibbstown, NJ, USA; DA, (–)-cocaine, antibodies for syn 1A, tyrosine hydroxylase, and polyhistidine, and all other reagents were from Sigma-Aldrich, St. Louis, MO, USA. Rats were purchased from

BoNT/C treatment of rat striatal tissue affects DA transport capacity and DAT phosphorylation

To examine the role of syn 1A on DAT function in neurons, we treated rat striatal tissue with BoNT/C and monitored effects on DA transport and DAT phosphorylation. For uptake experiments, striatal slices were treated with vehicle or BoNT/C followed by preparation of synaptosomes and analysis of [3H]DA transport activity or DAT and syn 1A levels (Fig. 1). Synaptosomes prepared from tissue treated with 100 ng/mL BoNT/C for 20, 40, or 60 min displayed [3H]DA uptake activity that was increased to

Discussion

In this study we found that treatment of rat striatal tissue with BoNT/C led to increased synaptosomal DA transport activity and decreased DAT phosphorylation, while heterologous co-expression of syn 1A with DAT caused reductions in transport capacity and transporter surface expression. In addition we found specific co-precipitation of syn 1A with DAT, demonstrating the presence of a direct or indirect interaction between the proteins. These findings suggest that in neurons, syn 1A interactions

Acknowledgments

This work was supported by DA13147, ND EPSCoR Award, and UND Faculty Seed Award (R.A.V.), F31 DA17520 (M.A.C.), and P20 RR016741 to the University of North Dakota from the INBRE program of the National Center for Research Resources, and ND EPSCoR through NSF EPS-0447679. We thank Steven Adkins for excellent technical assistance.

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    Current address: Department of Pathology, Dartmouth Medical School, Lebanon, NH 03756, USA.

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