The neuronal glycine transporter GLYT2 associates with membrane rafts: functional modulation by lipid environment (original) (raw)
2013
Glycine is the main inhibitory neurotransmitter in the caudal areas of the brain, spinal cord and retina and plays an important role in the processing of sensory and motor information. In addition, glycine exerts a positive modulation in the excitatory glutamatergic neurotransmission by acting as a necessary co-agonist of glutamate in NMDA receptors. Glycine-mediated neurotransmission is terminated by neurotransmitter reuptake by plasma membrane glycine transporters GlyT1 and GlyT2. Mutations in the GlyT2 gene (SLC6A5) cause an impairment of glycinergic neurotransmission that produces hyperekplexia in humans because the neurotransmitter cannot be recaptured back to the presynaptic terminal to refill synaptic vesicles. Many of these mutations impair the normal intracellular trafficking of GlyT2, e.g. the substitution Y705C (c.2114A→G) described by our laboratory. Given the importance of GlyT2 intracellular trafficking, we have analysed the molecular mecanisms underling GlyT2 endocytosis, describing the dependence on dynamin and clathrin pathway of the constitutive and PKC-induced internalization of the transporter. Moreover, we have described that GlyT2 internalizes constitutively associated to lipid rafts whereas in the PKC-induced endocytosis a previous lateral displacement to non-raft domains is needed before GlyT2 internalizes to endosomal compartments. In addition, we have described that ubiquitination in the GlyT2 C-terminal end plays an important role in both internalization process. Whereas PKC-induced endocytosis of GlyT2 needs the ubiquitination of a single lysine in position K791, constitutive endocytosis of the transporter needs the ubiquitination of a cluster of lysines in positions K751, K773 ,K787, K791. Simultaneous mutation of this c-ter lysine cluster reduced basal ubiquitination of the transporter and increased notably its half-life and stability. Also, we have described that GlyT2 ubiquitination status is highly sensitive to ubiquitin homeostasis in neurons, since the reduction of the free monomeric ubiquitin pool achieved by UCHL1 inhibition reduces significantly GlyT2 ubiquitination. Moreover, in these conditions constitutive endocytosis of the transporter is impaired, denoting the importance of the ubiquitination mechanism in the GlyT2 internalization process. In this work we have also described that Na/K ATPase is a new interacting partner for GlyT2. GlyT2 interacts with α catalytic subunits, especially with α3NKA subunit, and this interaction is restricted to lipid raft subdomains. α subunits bind ouabain, an endogenous cardiotonic steroid that induces the activation of many intracellular signaling events. In the presence of ouabain we have observed endocytosis and degradation of the lipid raft associated pool of GlyT2, which represents a drastic new pathway of transporter regulation. Further studies are needed to dilucidate the mechanisms underling this process, but these findings could help designing more effective actions in glycinergic neuromotor disorders including hyperekplexia and myoclonus, or other glycinergic-related dysfunctions as neuropathic pain or epilepsy.
Glycoprotein M6a as a signaling transducer in neuronal lipid rafts
Neuroscience Research, 2018
Neuronal development is composed of the complex steps which involve many signaling proteins. On the other hand, there are many proteins highly expressed in the differentiated neurons at developmental stages, but of which physiological functions are not precisely known so far. Glycoprotein 6a (GPM6a) currently belongs to such proteins. This protein has four-transmembrane domains and is a member of proteolipid protein family. Recently, we demonstrated that GPM6a is highly concentrated in lipid rafts of the developing neuron with its palmitoylation, and that this molecule is involved in rapid determination of the neuronal polarity, in response to laminin, an extracellular matrix protein (Honda et al., J Neurosci 37: 4046-64 [2017]). Since lipid rafts are membrane domains enriched in sphingolipids and cholesterol, have lower fluidity than other membrane areas and are believed to be signaling "hot-spots", and here, we discuss the functions of this protein in neuronal lipid raft signaling for neuronal development.
Journal of Biological Chemistry, 2001
Glycine transporter GLYT2 is an axonal glycoprotein involved in the removal of glycine from the synaptic cleft. To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putative N-glycosylation sites on the transport activity, intracellular traffic in COS cells, and asymmetrical distribution of this protein in polarized Madin-Darby canine kidney (MDCK) cells. Transport activity was reduced by 35-40% after enzymatic deglycosylation of the transporter reconstituted into liposomes.
Glycine transporters: essential regulators of synaptic transmission
Biochemical Society Transactions, 2006
Glycine is a major inhibitory neurotransmitter in the mammalian CNS (central nervous system). Glycinergic neurotransmission is terminated by the uptake of glycine into glycinergic nerve terminals and neighbouring glial cells. This uptake process is mediated by specific Na + /Cl − -dependent GlyTs (glycine transporters), GlyT1 and GlyT2. GlyT1, in addition, is thought to regulate the concentration of glycine at excitatory synapses containing NMDARs (N-methyl-d-aspartate receptors), which require glycine as a co-agonist. We have analysed the physiological roles and regulation of GlyT1 and GlyT2 by generating transporter-deficient mice and searching for interacting proteins. Our genetic results indicate that at glycinergic synapses, the glial transporter GlyT1 catalyses the removal of glycine from the synaptic cleft, whereas GlyT2 is required for the re-uptake of glycine into nerve terminals, thereby allowing for neurotransmitter reloading of synaptic vesicles. Both GlyT1 and GlyT2 are essential for CNS function, as revealed by the lethal phenotypes of the respective knockout mice. Mice expressing only a single GlyT1 allele are phenotypically normal but may have enhanced NMDAR function. GlyT2 is highly enriched at glycinergic nerve terminals, and Ca 2+ -triggered exocytosis and internalization are thought to regulate GlyT2 numbers in the pre-synaptic plasma membrane. We have identified different interacting proteins that may play a role in GlyT2 trafficking and/or pre-synaptic localization.
2005
Sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and certain signaling molecules segregate from bulk membrane lipids into lateral domains termed lipid rafts, which are often isolated based on their insolubility in cold nonionic detergents. During immunohistological studies of gangliosides, major sphingolipids of the brain, we found that cold Triton X-100 solubility is bidirectional, leading to histological redistribution from gray to white matter. When brain sections were treated with z0.25% Triton X-100 at 4 8C, ganglioside GD1a, which is normally enriched in gray matter and depleted in white matter, redistributed into white matter tracts. Incubation of brain sections from knockout mice lacking GD1a with wild-type sections in the presence of cold Triton X-100 resulted in GD1a redistribution from wild-type gray matter to knockout white matter. GM1, which is normally enriched in white matter, remained in white matter after cold detergent treatment and did not migrate to knockout mouse brain sections. However, when gray matter gangliosides were enzymatically converted into GM1 in situ, the newly formed GM1 transmigrated to knockout mouse brain sections in the presence of cold detergent. When purified GD1a was added to knockout mouse brain sections in the presence of cold Triton X-100, it preferentially incorporated into white matter tracts. These data demonstrate that brain white matter is a sink for gangliosides, which redistribute from gray matter in the presence of low concentrations of cold Triton X-100. A GPI-anchored protein, Thy-1, also transmigrated from wild-type to Thy-1 knockout mouse brain sections in the presence of detergent at 4 8C, although less efficiently than did gangliosides. These data raise technical challenges for using nonionic detergents in certain histological protocols and for isolation of lipid rafts from brain tissue. D
Biochemical Journal, 2008
The neuronal glycine transporter GLYT2 controls the availability of the neurotransmitter in glycinergic synapses, and the modulation of its function may influence synaptic transmission. The active transporter is located in membrane rafts and reaches the cell surface through intracellular trafficking. In the present study we prove that GLYT2 constitutively recycles between the cell interior and the plasma membrane by means of a monensin-sensitive trafficking pathway. Also, a regulated trafficking can be triggered by PMA. We demonstrate that PMA inhibits GLYT2 transport by causing net accumulation of the protein in internal compartments through an increase of the internalization rate. In addition, a small increase of plasma membrane delivery and a redistribution of the transporter to non-raft domains is triggered by PMA. A previously identified phorbol-ester-resistant mutant (K422E) displaying an acidic substitution in a regulatory site, exhibits constitutive traffic but, in contrast ...
The glycine transporter GLYT2 is a reliable marker for glycine-immunoreactive neurons
Molecular Brain Research, 1997
The glycine transporter GLYT2 is present in neurons of the spinal cord, the brain stem and the cerebellum. This localization is similar to that of glycine immunoreactivity, suggesting a causal relationship between GLYT2 expression and glycine distribution. In this report, we analyzed if such a relationship does exist by using neuronal cultures derived from embryonic spinal cord. GLYT2 was synthesized in a small subpopulation of neurons where it was targeted both to dendrites and to axons, being the axonal content higher than the dendritic one. At early stages in the development of cultured spinal neurons, the highest GLYT2 levels were found in the axonal growth cones. As the culture matured, immunoreactivity extended to the axonal shaft. Double-immunofluorescence experiments indicated a perfect co-localization of GLYT2 and glycine immunoreactivity in cultured neurons. Moreover, the concentration of glycine into neurons expressing GLYT2 was proportional to the concentration of the transporter. This observation was reproduced in GLYT2-transfected COS cells. These evidences indicate that the high content of glycine observed in some neurons in culture is indeed achieved by the concentrative task performed by GLYT2, and that GLYT2 can be used as a reliable marker for identification of glycine-enriched neurons. q 1997 Elsevier Science B.V.
Biochemical and Biophysical Research Communications, 2006
Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37°C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-b-cyclodextrin (mbCD) causes alteration in the buoyancy properties of proteins initially present in Brij-as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated. vesicle protein 2; SNAP25, synaptosomal-associated protein of 25 kDa; SNARE, soluble NSF-attachment protein receptor; VAMP, vesicle-associated membrane protein.
Journal of Neurochemistry, 2002
We studied by immunocytochemical localization, the glycine neurotransmitter transporter (GLYT2) in mouse brain, using polyclonal antibodies raised against recombinant N-terminus and loop fusion proteins. Western analysis and immunocytochemistry of mouse brain frozen sections revealed caudal-rostrai gradient of GLYT2 distribution with massive accumulation in the spinal cord, brainstem, and less in the cerebellum. Immunereactivity was detected in processes with varicosities but not cell bodies. A correlation was observed between the pattern we obtained and previously reported strychnine binding studies. The results indicate that GLYT2 is involved in the termination of glycine neurotransmission accompanying the glycine receptor at the classic inhibitory system in the hindbrain.