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Open Access Research

cGMP-dependent protein kinase Iα associates with the antidepressant-sensitive serotonin transporter and dictates rapid modulation of serotonin uptake

Jennifer A Steiner1, Ana Marin D Carneiro1, Jane Wright1, Heinrich JG Matthies2, Harish C Prasad1, Christian K Nicki3, Wolfgang R Dostmann3, Carrie C Buchanan1, Jackie D Corbin2, Sharron H Francis2 and Randy D Blakely145*

Author Affiliations

1 Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

2 Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

3 Departments of Pharmacology and Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, VT 05405, USA

4 Department of Psychiatry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

5 Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232, USA

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Molecular Brain 2009, 2:26  doi:10.1186/1756-6606-2-26

Published: 5 August 2009

Abstract

Background

The Na+/Cl--dependent serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is a critical element in neuronal 5-HT signaling, being responsible for the efficient elimination of 5-HT after release. SERTs are not only targets for exogenous addictive and therapeutic agents but also can be modulated by endogenous, receptor-linked signaling pathways. We have shown that neuronal A3 adenosine receptor activation leads to enhanced presynaptic 5-HT transport in vitro and an increased rate of SERT-mediated 5-HT clearance in vivo. SERT stimulation by A3 adenosine receptors derives from an elevation of cGMP and subsequent activation of both cGMP-dependent protein kinase (PKG) and p38 mitogen-activated protein kinase. PKG activators such as 8-Br-cGMP are known to lead to transporter phosphorylation, though how this modification supports SERT regulation is unclear.

Results

In this report, we explore the kinase isoform specificity underlying the rapid stimulation of SERT activity by PKG activators. Using immortalized, rat serotonergic raphe neurons (RN46A) previously shown to support 8-Br-cGMP stimulation of SERT surface trafficking, we document expression of PKGI, and to a lower extent, PKGII. Quantitative analysis of staining profiles using permeabilized or nonpermeabilized conditions reveals that SERT colocalizes with PKGI in both intracellular and cell surface domains of RN46A cell bodies, and exhibits a more restricted, intracellular pattern of colocalization in neuritic processes. In the same cells, SERT demonstrates a lack of colocalization with PKGII in either intracellular or surface membranes. In keeping with the ability of the membrane permeant kinase inhibitor DT-2 to block 8-Br-cGMP stimulation of SERT, we found that DT-2 treatment eliminated cGMP-dependent kinase activity in PKGI-immunoreactive extracts resolved by liquid chromatography. Similarly, treatment of SERT-transfected HeLa cells with small interfering RNAs targeting endogenous PKGI eliminated 8-Br-cGMP-induced regulation of SERT activity. Co-immunoprecipitation studies show that, in transporter/kinase co-transfected cells, PKGIα specifically associates with hSERT.

Conclusion

Our findings provide evidence of a physical and compartmentalized association between SERT and PKGIα that supports rapid, 8-Br-cGMP-induced regulation of SERT. We discuss a model wherein SERT-associated PKGIα supports sequentially the mobilization of intracellular transporter-containing vesicles, leading to enhanced surface expression, and the production of catalytic-modulatory SERT phosphorylation, leading to a maximal enhancement of 5-HT clearance capacity.