Characterization of Strychnine-sensitive Glycine Receptor in the Intact Frog Retina: Modulation by Protein Kinases

Glycine receptors (GlyRs) are important mediators of fast inhibitory neurotransmission in the mammalian central nervous system. Their function is controlled by multiple cellular mechanisms, including intracellular regulatory processes. Modulation of GlyR function by protein kinases has been reported for many cell types, involving different techniques, and often yielding contradictory results. Here, we studied the effects of protein kinase C (PKC) and cAMP-dependent protein kinase A (PKA) on glycine induced currents in HEK293 cells expressing human homomeric α1 and heteromeric α1-β GlyRs using whole-cell patch clamp techniques as well as internalization assays. In whole-cell patch-clamp measurements, modulators were applied in the intracellular buffer at concentrations between 0.1 μM and 0.5 μM. EC50 of glycine increased upon application of the protein kinase activators Forskolin and phorbol-12-myristate-13-acetate (PMA) but decreased in the presence of the PKC inhibitor Staurosporine aglycon and the PKA inhibitor H-89. Desensitization of recombinant α1 receptors was significantly increased in the presence of Forskolin. Staurosporine aglycon, on the other hand decreased desensitization of heteromeric α1-β GlyRs. The time course of receptor activation was determined for homomeric α1 receptors and revealed two simultaneous effects: cells showed a decrease of EC50 after 3–6 min of establishing whole-cell configuration. This effect was independent of protein kinase modulators. All modulators of PKA and PKC, however, produced an additional shift of EC50, which overlay and eventually exceeded the cells intrinsic variation of EC50. The effect of kinase activators was abolished if the corresponding inhibitors were co-applied, consistent with PKA and PKC directly mediating the modulation of GlyR function. Direct effects of PKA- and PKC-modulators on receptor expression on transfected HEK cells were monitored within 15 min of drug application, showing a significant increase of receptor internalization with PKA and PKC activators, while the corresponding inhibitors had no significant effect on receptor surface expression or internalization. Our results confirm the observation that phosphorylation via PKA and PKC has a direct effect on the GlyR ion channel complex and plays an important role in the fine-tuning of glycinergic signaling.

Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 89%

PKA and PKC Modulators Affect Ion Channel Function and Internalization of Recombinant Alpha1 and Alpha1-Beta Glycine Receptors

Breitinger

Bahnassawy

Janzen

et al. 2018

“…Downstream events of such factor(s) could at least imply protein kinase activity. In that respect, it has been shown that, in the retina and in auditory nuclei, protein kinases modulate strychnine binding and, hence, GlyR expression (Salceda and Aguirre-Ramirez, 2005; Yan et al, 2007). Figure 1 summarizes the current findings on glycine and GlyR signalling in macroglial cells.…”

Section: Neuroglial Cellsmentioning

confidence: 99%

Glycine and glycine receptor signalling in non-neuronal cells

Eynden

2009

Glycine is an inhibitory neurotransmitter acting mainly in the caudal part of the central nervous system. Besides this neurotransmitter function, glycine has cytoprotective and modulatory effects in different non-neuronal cell types. Modulatory effects were mainly described in immune cells, endothelial cells and macroglial cells, where glycine modulates proliferation, differentiation, migration and cytokine production. Activation of glycine receptors (GlyRs) causes membrane potential changes that in turn modulate calcium flux and downstream effects in these cells. Cytoprotective effects were mainly described in renal cells, hepatocytes and endothelial cells, where glycine protects cells from ischemic cell death. In these cell types, glycine has been suggested to stabilize porous defects that develop in the plasma membranes of ischemic cells, leading to leakage of macromolecules and subsequent cell death. Although there is some evidence linking these effects to the activation of GlyRs, they seem to operate in an entirely different mode from classical neuronal subtypes.

“…Sarcosine (aka N-methylglycine), is a glycine transporter 1 (GlyT1) inhibitor that can increase extracellular glycine (Lopez-Corcuera et al, 1998;Herdon et al, 2001) via blockade of GlyT1-mediated glycine uptake or through a hetero-exchange of sarcosine for glycine (Herdon et al, 2001). Sarcosine agonizes the glycine receptor (GlyR) via binding to its strychnine-sensitive site (Salceda and Aguirre-Ramirez, 2005;Zhang et al, 2009). In addition, sarcosine acts as a co-agonist of N-methyl-D-aspartate receptor (NMDAR) (Ryley Parrish et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

Sarcosine Suppresses Epileptogenesis in Rats With Effects on Hippocampal DNA Methylation

Shen

Weltha

Cook

et al. 2020

Epileptogenesis is a common consequence of brain insults, however, the prevention or delay of the epileptogenic process remains an important unmet medical challenge. Overexpression of glycine transporter 1 (GlyT1) is proposed as a pathological hallmark in the hippocampus of patients with temporal lobe epilepsy (TLE), and we previously demonstrated in rodent epilepsy models that augmentation of glycine suppressed chronic seizures and altered acute seizure thresholds. In the present study we evaluated the effect of the GlyT1 inhibitor, sarcosine (aka N-methylglycine), on epileptogenesis and also investigated possible mechanisms. We developed a modified rapid kindling model of epileptogenesis in rats combined with seizure score monitoring to evaluate the antiepileptogenic effect of sarcosine. We used immunohistochemistry and Western blot analysis for the evaluation of GlyT1 expression and epigenetic changes of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the epileptogenic hippocampi of rats, and further evaluated expression changes in enzymes involved in the regulation of DNA methylation, ten-eleven translocation methylcytosine dioxygenase 1 (TET1), DNA-methyltransferase 1 (DNMT1), and DNMT3a. Our results demonstrated: (i) experimental evidence that sarcosine (3 g/kg, i.p. daily) suppressed kindling epileptogenesis in rats; (ii) the sarcosine-induced antiepileptogenic effect was accompanied by a suppressed hippocampal GlyT1 expression as well as a reduction of hippocampal 5mC levels and a corresponding increase in 5hmC; and (iii) sarcosine treatment caused differential expression changes of TET1 and DNMTs. Together, these findings suggest that sarcosine has unprecedented disease-modifying properties in a kindling model of epileptogenesis in rats, which was associated with altered hippocampal DNA methylation. Thus, manipulation of the glycine system is a potential therapeutic approach to attenuate the development of epilepsy.