P2Y Purinergic Regulation of the Glycine Neurotransmitter Transporters

Abstract

The sodium-and chloride-coupled glycine neurotransmitter transporters (GLYTs) control the availability of glycine at glycine-mediated synapses. The mainly glial GLYT1 is the key regulator of the glycine levels in glycinergic and glutamatergic pathways, whereas the neuronal GLYT2 is involved in the recycling of synaptic glycine from the inhibitory synaptic cleft. In this study, we report that stimulation of P2Y purinergic receptors with 2-methylthioadenosine 5 -diphosphate in rat brainstem/spinal cord primary neuronal cultures and adult rat synaptosomes leads to the inhibition of GLYT2 and the stimulation of GLYT1 by a paracrine regulation. These effects are mainly mediated by the ADP-preferring subtypes P2Y 1 and P2Y 13 because the effects are partially reversed by the specific antagonists N 6 -methyl-2 -deoxyadenosine-3 ,5 -bisphosphate and pyridoxal-5 -phosphate-6-azo(2-chloro-5-nitrophenyl)-2,4-disulfonate and are totally blocked by suramin. P2Y 12 receptor is additionally involved in GLYT1 stimulation. Using pharmacological approaches and siRNAmediated protein knockdown methodology, we elucidate the molecular mechanisms of GLYT regulation. Modulation takes place through a signaling cascade involving phospholipase C activation, inositol 1,4,5-trisphosphate production, intracellular Ca 2؉ mobilization, protein kinase C stimulation, nitric oxide formation, cyclic guanosine monophosphate production, and protein kinase G-I (PKG-I) activation. GLYT1 and GLYT2 are differentially sensitive to NO/cGMP/PKG-I both in brain-derived preparations and in heterologous systems expressing the recombinant transporters and P2Y 1 receptor. Sensitivity to 2-methylthioadenosine 5 -diphosphate by GLYT1 and GLYT2 was abolished by small interfering RNA (siRNA)-mediated knockdown of nitric-oxide synthase. Our data may help define the role of GLYTs in nociception and pain sensitization.

Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia

“…The Myc-Sec24D and HAGlyT2 constructs in the pcDNA3 plasmid (Invitrogen) were generously provided by Francisco Zafra (Centro de Biología Molecular Severo Ochoa). GlyT2 (28) was subcloned into pcDNA3 or pRFP (BglII-SalI sites), and the GlyT2 mutants were constructed by site-directed mutagenesis using the QuikChange kit (Stratagene) (29). S512R was subcloned in the unique EcoRI and XbaI sites flanking HA-GlyT2.…”

A Novel Dominant Hyperekplexia Mutation Y705C Alters Trafficking and Biochemical Properties of the Presynaptic Glycine Transporter GlyT2

“…7B). This region cannot currently be accurately modeled because of significant divergence in sequence and length compared with LeuT Aa (18,21). However, equivalent conserved cysteines have been shown to form a disulfide bond in other members of the SLC6 family (30,31).…”

“…Cell Surface Labeling-Thiol-specific biotinylation and total surface biotinylation was performed with MTSEA-biotin (Toronto Research Chemicals Inc., Toronto, Canada) and sulfo-NHS-SS-biotin (Pierce) on transfected COS7 cells as described (20,21). 0.5 mM MTSEA-biotin or 1.5 mg/ml sulfo-NHS-SS-biotin and a 3-h incubation with streptavidin-agarose beads (Sigma) were used.…”

Glycine transporters type 1 inhibitor promotes brain preconditioning against NMDA-induced excitotoxicity

“…GlyT1 protein is associated to glutamatergic synapses where its main function is to control the level of glycine (Zafra et al, 1995a;Zafra et al, 1995b;Cubelos et al, 2005;Jim enez et al, 2011;Vargas-Medrano et al, 2011). The inhibition of GlyT1 promotes an increase on glycine levels in synaptic terminals, which potentiates the NMDAR activation (Bergeron et al, 1998;Chen et al, 2003;Kinney et al, 2003;Lim et al, 2004;Zhang et al, 2009).…”