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Colocalization and regulated physical association of presynaptic serotonin transporters with A₃ adenosine receptors.

ABSTRACT: Activation of A₃ adenosine receptors (A₃ARs) rapidly enhances the activity of antidepressant-sensitive serotonin (5-HT) transporters (SERTs) in vitro, ex vivo, and in vivo. A₃AR agonist stimulation of SERT activity is lost in A₃AR knockout mice. A₃AR-stimulated SERT activity is mediated by protein kinase G1 (PKGI)- and p38 mitogen-activated protein kinase (MAPK)-linked pathways that support, respectively, enhanced SERT surface expression and catalytic activation. The mechanisms by which A₃ARs target SERTs among other potential effectors is unknown. Here we present evidence that A₃ARs are coexpressed with SERT in midbrain serotonergic neurons and form a physical complex in A₃AR/hSERT cotransfected cells. Treatment of A₃AR/SERT-cotransfected Chinese hamster ovary cells with the A₃AR agonist N⁶-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (1 μM, 10 min), conditions previously reported to increase SERT surface expression and 5-HT uptake activity, enhanced the abundance of A₃AR/SERT complexes in a PKGI-dependent manner. Cotransfection of SERT with L90V-A₃AR, a hyperfunctional coding variant identified in subjects with autism spectrum disorder, resulted in a prolonged recovery of receptor/transporter complexes after A₃AR activation. Because PKGI and nitric-oxide synthetase are required for A₃AR stimulation of SERT activity, and proteins PKGI and NOS both form complexes with SERT, our findings suggest a mechanism by which signaling pathways coordinating A₃AR signaling to SERT can be spatially restricted and regulated, as well as compromised by neuropsychiatric disorders.

SUBMITTER: Zhu CB

PROVIDER: S-EPMC3164334 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Colocalization and regulated physical association of presynaptic serotonin transporters with A₃ adenosine receptors.

Zhu Chong-Bin CB Lindler Kathryn M KM Campbell Nicholas G NG Sutcliffe James S JS Hewlett William A WA Blakely Randy D RD

Molecular pharmacology 20110624 3

Activation of A₃ adenosine receptors (A₃ARs) rapidly enhances the activity of antidepressant-sensitive serotonin (5-HT) transporters (SERTs) in vitro, ex vivo, and in vivo. A₃AR agonist stimulation of SERT activity is lost in A₃AR knockout mice. A₃AR-stimulated SERT activity is mediated by protein kinase G1 (PKGI)- and p38 mitogen-activated protein kinase (MAPK)-linked pathways that support, respectively, enhanced SERT surface expression and catalytic activation. The mechanisms by which A₃ARs ta ...[more]

PMID: 21705486

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Project description:Background and purposeBoth cannabinoid CB1 and adenosine A2A receptors (CB1 receptors and A2A receptors) control synaptic transmission at corticostriatal synapses, with great therapeutic importance for neurological and psychiatric disorders. A postsynaptic CB1 -A2A receptor interaction has already been elucidated, but the presynaptic A2A receptor-mediated control of presynaptic neuromodulation by CB1 receptors remains to be defined. Because the corticostriatal terminals provide the major input to the basal ganglia, understanding the interactive nature of converging neuromodulation on them will provide us with novel powerful tools to understand the physiology of corticostriatal synaptic transmission and interpret changes associated with pathological conditions.Experimental approachPharmacological manipulation of CB1 and A2A receptors was carried out in brain nerve terminals isolated from rats and mice, using flow synaptometry, immunoprecipitation, radioligand binding, ATP and glutamate release measurement. Whole-cell patch-clamp recordings were made in horizontal corticostriatal slices.Key resultsFlow synaptometry showed that A2A receptors were extensively co-localized with CB1 receptor-immunopositive corticostriatal terminals and A2A receptors co-immunoprecipitated CB1 receptors in these purified terminals. A2A receptor activation decreased CB1 receptor radioligand binding and decreased the CB1 receptor-mediated inhibition of high-K(+) -evoked glutamate release in corticostriatal terminals. Accordingly, A2A receptor activation prevented CB1 receptor-mediated paired-pulse facilitation and attenuated the CB1 receptor-mediated inhibition of synaptic transmission in glutamatergic synapses of corticostriatal slices.Conclusions and implicationsActivation of presynaptic A2A receptors dampened CB1 receptor-mediated inhibition of corticostriatal terminals. This constitutes a thus far unrecognized mechanism to modulate the potent CB1 receptor-mediated presynaptic inhibition, allowing frequency-dependent enhancement of synaptic efficacy at corticostriatal synapses.

Project description:The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] modulates many key brain functions including those subserving sensation, emotion, reward, and cognition. Efficient clearance of 5-HT after release is achieved by the antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). To identify novel SERT regulators, we pursued a proteomic analysis of mouse midbrain SERT complexes, evaluating findings in the context of prior studies that established a SERT-linked transcriptome. Remarkably, both efforts converged on a relationship of SERT with the synaptic adhesion protein neuroligin 2 (NLGN2), a post-synaptic partner for presynaptic neurexins, and a protein well-known to organize inhibitory GABAergic synapses. Western blots of midbrain reciprocal immunoprecipitations confirmed SERT/NLGN2 associations, and also extended to other NLGN2 associated proteins [e.g., α-neurexin (NRXN), gephyrin]. Midbrain SERT/NLGN2 interactions were found to be Ca(2+)-independent, supporting cis vs. trans-synaptic interactions, and were absent in hippocampal preparations, consistent with interactions arising in somatodendritic compartments. Dual color in situ hybridization confirmed co-expression of Tph2 and Nlgn2 mRNA in the dorsal raphe, with immunocytochemical studies confirming SERT:NLGN2 co-localization in raphe cell bodies but not axons. Consistent with correlative mRNA expression studies, loss of NLGN2 expression in Nlgn2 null mice produced significant reductions in midbrain and hippocampal SERT expression and function. Additionally, dorsal raphe 5-HT neurons from Nlgn2 null mice exhibit reduced excitability, a loss of GABAA receptor-mediated IPSCs, and increased 5-HT1A autoreceptor sensitivity. Finally, Nlgn2 null mice display significant changes in behaviors known to be responsive to SERT and/or 5-HT receptor manipulations. We discuss our findings in relation to the possible coordination of intrinsic and extrinsic regulation afforded by somatodendritic SERT:NLGN2 complexes.

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Colocalization and regulated physical association of presynaptic serotonin transporters with A₃ adenosine receptors.

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Colocalization and regulated physical association of presynaptic serotonin transporters with A₃ adenosine receptors.

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