Calcium-Dependent Regulation of the Neuronal Glycine Transporter GlyT2 by M2 Muscarinic Acetylcholine Receptors.

The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission and plays a key role in regulating nociceptive signal progression. The cholinergic system acting through muscarinic acetylcholine receptors (mAChRs) also mediates important regulations of nociceptive transmission being the M2 subtype the most abundantly expressed in the spinal cord. Here we studied the effect of M2 mAChRs stimulation on GlyT2 function co-expressed in a heterologous system with negligible levels of muscarinic receptor activity. We found GlyT2 is down-regulated by carbachol in a calcium-dependent manner. Different components involved in cell calcium homeostasis were analysed to establish a role in the mechanism of GlyT2 inhibition. GlyT2 down-regulation by carbachol was increased by thapsigargin and reduced by internal store depletion, although calcium release from endoplasmic reticulum or mitochondria had a minor role on GlyT2 inhibition. Our results are consistent with a GlyT2 sensitivity to intracellular calcium mobilized by M2 mAChRs in the subcortical area of the plasma membrane. A crucial role of the plasma membrane sodium calcium exchanger NCX is proposed.

Prehabilitation in patients undergoing pancreaticoduodenectomy: a randomized controlled trial.

INTRODUCTION: prehabilitation has been proposed as an effective tool to prevent postoperative complications in patients undergoing major abdominal surgery. However, no studies have demonstrated its effectiveness in pancreatic surgical patients. The aim of this study was to assess the impact of prehabilitation on postoperative complications in patients undergoing a pancreaticoduodenectomy (PD). METHODS: this was a randomized controlled trial. Eligible candidates who accepted to participate were randomized to the control (standard care) or intervention (standard care + prehabilitation) group. All patients with pancreatic or periampullary tumors who were candidates for pancreaticoduodenectomy were included. Patients who received neoadjuvant treatment were excluded. Prehabilitation covered three actions: a) nutritional support; b) control of diabetes and exocrine pancreatic insufficiency; and c) physical and respiratory training. The main study outcome was the proportion of patients who suffered postoperative complications. Secondary outcomes included the occurrence of specific complications (pancreatic leak and delayed gastric emptying) and hospital stay. RESULTS: forty patients were included in the analysis. Twenty-two patients were randomized to the control arm and 18, to the intervention group. No statistically significant differences were observed in terms of overall and major complications between the prehabilitation and standard care groups. Pancreatic leak was not statistically different between the groups (11% vs 27%, p = 0.204). However, DGE was significantly lower in the prehabilitation group (5.6% vs 40.9% in the standard care group, p = 0.01). CONCLUSION: prehabilitation did not reduce postoperative complications following pancreaticoduodenectomy. However, a reduction in DGE was observed. Further studies are needed to validate the role and the timing of prehabilitation in high-risk patients.

Preliminary results of a screening program for anal cancer and its precursors for HIV-infected men who have sex with men in Vigo-Spain.

BACKGROUND: Men who have sex with men (MSM) infected with human immunodeficiency virus (HIV) have the highest risk of developing anal cancer (AC). The objective of this study was to describe our screening implementation program in this population, and report the prevalence of human papillomavirus (HPV) anal infection, and cytological and histological findings in a Spanish medium-size community (Vigo, Spain). METHOD: Prospective cohort analysis of 240 HIV-infected MSM. Cellular anal sample and high risk HPV (HR-HPV)-tests were performed to study cytological changes and HPV genotyping. High resolution anoscopy (HRA) was performed in 209 patients. Results were analyzed with respect to epidemiological, clinical and analytical factors. RESULTS: Of 209 patients selected for HRA, the prevalence of HR-HPV anal infection, cytological and histological alterations was 85.6%, 47.5%, and 39.8%, respectively. Sensitivity and specificity for ≥ ASCUS (atypia of squamous cells of undetermined significance) cytology in relation to histological alterations were 61% and 85%, (OR: 8.7; IC 95%: 4.4-17.2), respectively. Observed concordance between high-grade squamous intraepithelial lesion (HSIL) cytology and HSIL anal intraepithelial neoplasia types 2 and 3 (AIN-2/3) histology was 64% (OR: 11.4; IC 95%: 3.6-36.7). One patient with HSIL cytology presented a prevalent anal squamous carcinoma. CONCLUSIONS: HRA was feasible with similar results to relevant groups. There was a high prevalence of anal HR-HPV infection, and cytological and histological alterations.

Presynaptic control of glycine transporter 2 (GlyT2) by physical and functional association with plasma membrane Ca2+-ATPase (PMCA) and Na+-Ca2+ exchanger (NCX).

Fast inhibitory glycinergic transmission occurs in spinal cord, brainstem, and retina to modulate the processing of motor and sensory information. After synaptic vesicle fusion, glycine is recovered back to the presynaptic terminal by the neuronal glycine transporter 2 (GlyT2) to maintain quantal glycine content in synaptic vesicles. The loss of presynaptic GlyT2 drastically impairs the refilling of glycinergic synaptic vesicles and severely disrupts neurotransmission. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans. Here, we show a novel endogenous regulatory mechanism that can modulate GlyT2 activity based on a compartmentalized interaction between GlyT2, neuronal plasma membrane Ca(2+)-ATPase (PMCA) isoforms 2 and 3, and Na(+)/Ca(2+)-exchanger 1 (NCX1). This GlyT2·PMCA2,3·NCX1 complex is found in lipid raft subdomains where GlyT2 has been previously found to be fully active. We show that endogenous PMCA and NCX activities are necessary for GlyT2 activity and that this modulation depends on lipid raft integrity. Besides, we propose a model in which GlyT2·PMCA2-3·NCX complex would help Na(+)/K(+)-ATPase in controlling local Na(+) increases derived from GlyT2 activity after neurotransmitter release.

Na+/K+-ATPase is a new interacting partner for the neuronal glycine transporter GlyT2 that downregulates its expression in vitro and in vivo.

The neuronal glycine transporter GlyT2 plays a fundamental role in the glycinergic neurotransmission by recycling the neurotransmitter to the presynaptic terminal. GlyT2 is the main supplier of glycine for vesicle refilling, a process that is absolutely necessary to preserve quantal glycine content in synaptic vesicles. Alterations in GlyT2 activity modify glycinergic neurotransmission and may underlie several neuromuscular disorders, such as hyperekplexia, myoclonus, dystonia, and epilepsy. Indeed, mutations in the gene encoding GlyT2 are the main presynaptic cause of hyperekplexia in humans and produce congenital muscular dystonia type 2 (CMD2) in Belgian Blue cattle. GlyT2 function is strictly coupled to the sodium electrochemical gradient actively generated by the Na+/K+-ATPase (NKA). GlyT2 cotransports 3Na+/Cl-/glycine generating large rises of Na+ inside the presynaptic terminal that must be efficiently reduced by the NKA to preserve Na+ homeostasis. In this work, we have used high-throughput mass spectrometry to identify proteins interacting with GlyT2 in the CNS. NKA was detected as a putative candidate and through reciprocal coimmunoprecipitations and immunocytochemistry analyses the association between GlyT2 and NKA was confirmed. NKA mainly interacts with the raft-associated active pool of GlyT2, and low and high levels of the specific NKA ligand ouabain modulate the endocytosis and total expression of GlyT2 in neurons. The ouabain-mediated downregulation of GlyT2 also occurs in vivo in two different systems: zebrafish embryos and adult rats, indicating that this NKA-mediated regulatory mechanism is evolutionarily conserved and may play a relevant role in the physiological control of inhibitory glycinergic neurotransmission.

A novel dominant hyperekplexia mutation Y705C alters trafficking and biochemical properties of the presynaptic glycine transporter GlyT2.

Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this orphan disorder can have serious consequences, including sudden infant death. Dominant and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1) are the major cause of this disorder. However, recessive mutations in the presynaptic Na(+)/Cl(-)-dependent glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of startle disease. In this study, systematic DNA sequencing of SLC6A5 revealed a new dominant GlyT2 mutation: pY705C (c.2114A→G) in transmembrane domain 11, in eight individuals from Spain and the United Kingdom. Curiously, individuals harboring this mutation show significant variation in clinical presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal respiration, facial dysmorphism, delayed motor development, or intellectual disability. We functionally characterized this mutation using molecular modeling, electrophysiology, [(3)H]glycine transport, cell surface expression, and cysteine labeling assays. We found that the introduced cysteine interacts with the cysteine pair Cys-311-Cys-320 in the second external loop of GlyT2. This interaction impairs transporter maturation through the secretory pathway, reduces surface expression, and inhibits transport function. Additionally, Y705C presents altered H(+) and Zn(2+) dependence of glycine transport that may affect the function of glycinergic neurotransmission in vivo.

An aspartate residue in the external vestibule of GLYT2 (glycine transporter 2) controls cation access and transport coupling.

Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na+-Cl--glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na+. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na+ interactions. Replacement of Asp471 in this region reduced Na+ affinity and Na+ co-operativity of transport, an effect not produced in the homologous position (Asp295) in GLYT1. Unlike the GLYT1-Asp295 mutation, this Asp471 mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [3H]glycine accumulation. The homologous Asp471 and Asp295 positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na+ and Li+-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp471 in controlling cation access to GLYT2 Na+ sites, ion coupling during transport and the subsequent conformational changes.

Evaluation of the Insecticidal Potential of Heterotheca inuloides Acetonic and Methanolic Extracts against Spodoptera frugiperda and Their Ecotoxicological Effect on Poecilia reticulata.

For the management of Spodoptera frugiperda, botanical extracts have been used to reduce the environmental impacts of synthetic chemical pesticides. In the present investigation, the insecticidal activity of the acetonic and methanolic extracts of Heterotheca inuloides (Asteraceae) and of the main compound 7-hydroxy-3,4-dihydrocadalene on this pest as well as its ecotoxicological effect on Poecilia reticulata were evaluated. A greater insecticidal response was obtained from the acetonic extracts than from the methanolic extracts, with LC50 values of 730.4 ppm and 711.7 ppm for samples 1 and 2, respectively. Similarly, there was a lethal effect on 50% of the P. reticulata population at low concentrations in the acetonic extract compared to the methanolic extract. The sesquiterpene 7-hydroxy-3,4-dihydrocadalene has greater insecticidal activity by presenting an LC50 of 44.36 ppm; however, it is classified as moderately toxic for guppy fish.

[Vacuum assisted closure in open abdomen and deferred closure: experience in 23 patients]. / Vacuum assisted closure: utilidad en el abdomen abierto y cierre diferido. Experiencia en 23 pacientes.

INTRODUCTION: We analyse our experience and the results obtained with the use of vacuum assisted closure (VAC(®), KCI Clinic Spain SL) in the management of open abdomen. MATERIAL AND METHODS: We retrospectively reviewed the laparostomies performed between June 2006 and March 2011 using VAC(®) treatment in the Hospital Xeral-Cíes, Vigo. RESULTS: We included 23 consecutive patients (18 males and 5 females) on whom the VAC(®) was used in the open abdomen due to different indications (abdominal trauma, peritonitis, pancreatitis, ischaemic disease or abdominal compartmental syndrome). The VAC(®) needed changing a mean of 3.1 times per patient (range 1-7), with total mean treatment duration of 14.8 days (2-43) until closure, primary closure being achieved in 18 out of 21 patients (86%). The mean hospital stay was 110.1 days (8-163) and 6 patients (26%) died during their hospital stay due to problems related to their underlying disease. Seven cases (30%) had complications during the VAC® therapy: 3 intra-abdominal abscesses (13%), 4 fistulas or suture dehiscence (17%), and 1 evisceration (4%). CONCLUSIONS: VAC(®) therapy is simple to manage, with an acceptable rate of complication, particularly of intestinal fistulas, and a reduced mortality. Of the various systems available for the deferred closure of the abdomen, the VAC(®) has made considerable progress in the past few years, mainly due to its adaptable material, and its numerous advantages. Its use will possibly increase in the future.

Effect of COVID-19 on quality of life of persons aged >70 years with adult spinal deformity: A cross-sectional case-control study.

This observational, cross-sectional case-control study evaluates the impact of coronavirus disease 2019 (COVID-19) on health-related quality of life (HRQoL) in elderly persons who have undergone surgery for adult spinal deformity (ASD). On December 31, 2019, the Chinese authorities first reported severe acute respiratory syndrome coronavirus 2, and on March 11, 2020, it was declared a pandemic. The pandemic seems to have had a negative effect on elderly patients who underwent ASD, in terms of functional and psychological quality of life. We selected patients with ASD aged > 70 years who had undergone surgery between 2010 and 2015 and compared them with age- and sex-matched patients who did not have ASD. We recorded sociodemographic variables, type of surgery, levels of spinal fusion, HRQoL (Scoliosis Research Society-22, Short Form 12 Health Survey, EuroQol-5D [EQ-5], Geriatric Depression Scale [Yesavage] [GDS], Modified Frailty Index-11, and Barthel index), fear of visiting a health center, fear of leaving one's house, and adherence to preventive measures. The study population comprised 174 patients (mean [standard deviation] age, 77.3 [5.9] years; 86% women), of whom 87 had undergone surgery for ASD. The incidence of COVID-19 was higher in patients aged > 85 years (P = .041), urban areas (P = .047), and in patients in long-term care (P = .03). Similarly, no differences were observed for the ability to cope with the pandemic (P > .05). Patients who underwent surgery also had a higher risk of depression (GDS, 6.7 [P = .02]), a lower EQ-5 score (P = .001), a higher body mass index (P = .004), greater consumption of drugs (P < .001), especially opiates (P < .001). Patients who underwent surgery constitute a vulnerable population during the COVID-19 pandemic, with poorer quality of life and had a much higher risk of depression. They are also polymedicated and prefrail, adhere well to COVID-19 preventive measures, and do not seem to fear visiting health centers.

Subcellular localization of the neuronal glycine transporter GLYT2 in brainstem.

The neuronal glycine transporter GLYT2 belongs to the neurotransmitter:sodium:symporter (NSS) family and removes glycine from the synaptic cleft, thereby aiding the termination of the glycinergic signal and achieving the reloading of the presynaptic terminal. The task fulfilled by this transporter is fine tuned by regulating both transport activity and intracellular trafficking. Different stimuli such as neuronal activity or protein kinase C (PKC) activation can control GLYT2 surface levels although the intracellular compartments where GLYT2 resides are largely unknown. Here, by biochemical and immunological techniques in combination with electron and confocal microscopy, we have investigated the subcellular distribution of GLYT2 in rat brainstem tissue, and characterized the vesicles that contain the transporter. GLYT2 is shown to be present in small and larger vesicles that contain the synaptic vesicle protein synaptophysin, the recycling endosome small GTPase Rab11, and in the larger vesicle population, the vesicular inhibitory amino acid transporter VIAAT. Rab5A, the GABA transporter GAT1, synaptotagmin2 and synaptobrevin2 (VAMP2) were not present. Coexpression of a Rab11 dominant negative mutant with recombinant GLYT2 impaired transporter trafficking and glycine transport. Dual immunogold labeling of brainstem synaptosomes showed a very close proximity of GLYT2 and Rab11. Therefore, the intracellular GLYT2 resides in a subset of endosomal membranes and may traffic around several compartments, mainly Rab11-positive endosomes.

Clinical patterns and outcomes of ischaemic colitis: results of the Working Group for the Study of Ischaemic Colitis in Spain (CIE study).

BACKGROUND: There is a lack of prospective studies evaluating the natural history of colonic ischaemia (CI). We performed such a study to evaluate the clinical presentation, outcome, and mortality as well as clinical variables associated with poor prognosis. METHODS: An open, prospective, and multicentre study was conducted in 24 Spanish hospitals serving a population of 3.5 million people. The study included only patients who met criteria for definitive or probable CI. A website (www.colitisisquemica.org) provided logistical support. RESULTS: A total of 364 patients met criteria for inclusion. CI was suspected clinically in only 24.2% of cases. The distribution of clinical patterns was as follows: reversible colopathy (26.1%), transient colitis (43.7%), gangrenous colitis (9.9%), fulminant pancolitis (2.5%), and chronic segmental colitis (17.9%). A total of 47 patients (12.9%) had an unfavorable outcome as defined by mortality and/or the need for surgery. Multivariate analysis identified the following signs as independent risk factors for an unfavorable outcome: abdominal pain without rectal bleeding [odds ratio (OR) 3.9; 95% confidence interval (CI) = 1.6-9.3], non-bloody diarrhoea (OR 10; 95% CI = 3.7-27.4), and peritoneal signs (OR 7.3; 95% CI = 2.7-19.6). Unfavorable outcomes also were more frequent in isolated right colon ischaemia (IRCI) compared with non-IRCI (40.9 vs. 10.3%, respectively; p < 0.0001). The overall mortality rate was 7.7%. CONCLUSIONS: The clinical presentation of CI is very heterogeneous, perhaps explaining why clinical suspicion of this disease is so low. The presence of IRCI, and occurrence of peritoneal signs or onset of CI as severe abdominal pain without bleeding, should alert the physician to a potentially unfavorable course.

Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543.

The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission by controlling the extracellular concentration of synaptic glycine and the supply of neurotransmitter to the presynaptic terminal. Spinal cord glycinergic neurons present in the dorsal horn diminish their activity in pathological pain conditions and behave as gate keepers of the touch-pain circuitry. The pharmacological blockade of GlyT2 reduces the progression of the painful signal to rostral areas of the central nervous system by increasing glycine extracellular levels, so it has analgesic action. O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-l-serine (ALX1393) and N-[[1-(dimethylamino)cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide (ORG25543) are two selective GlyT2 inhibitors with nanomolar affinity for the transporter and analgesic effects in pain animal models, although with deficiencies which preclude further clinical development. In this report, we performed a comparative ligand docking of ALX1393 and ORG25543 on a validated GlyT2 structural model including all ligand sites constructed by homology with the crystallized dopamine transporter from Drosophila melanogaster. Molecular dynamics simulations and energy analysis of the complex and functional analysis of a series of point mutants permitted to determine the structural determinants of ALX1393 and ORG25543 discrimination by GlyT2. The ligands establish simultaneous contacts with residues present in transmembrane domains 1, 3, 6, and 8 and block the transporter in outward-facing conformation and hence inhibit glycine transport. In addition, differential interactions of ALX1393 with the cation bound at Na1 site and ORG25543 with TM10 define the differential sites of the inhibitors and explain some of their individual features. Structural information about the interactions with GlyT2 may provide useful tools for new drug discovery.

The presynaptic glycine transporter GlyT2 is regulated by the Hedgehog pathway in vitro and in vivo.

The identity of a glycinergic synapse is maintained presynaptically by the activity of a surface glycine transporter, GlyT2, which recaptures glycine back to presynaptic terminals to preserve vesicular glycine content. GlyT2 loss-of-function mutations cause Hyperekplexia, a rare neurological disease in which loss of glycinergic neurotransmission causes generalized stiffness and strong motor alterations. However, the molecular underpinnings controlling GlyT2 activity remain poorly understood. In this work, we identify the Hedgehog pathway as a robust controller of GlyT2 expression and transport activity. Modulating the activation state of the Hedgehog pathway in vitro in rodent primary spinal cord neurons or in vivo in zebrafish embryos induced a selective control in GlyT2 expression, regulating GlyT2 transport activity. Our results indicate that activation of Hedgehog reduces GlyT2 expression by increasing its ubiquitination and degradation. This work describes a new molecular link between the Hedgehog signaling pathway and presynaptic glycine availability.

Trafficking properties and activity regulation of the neuronal glycine transporter GLYT2 by protein kinase C.

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 with the wild-type, fails to show glycine uptake inhibition, membrane raft redistribution and trafficking modulation by PMA. We prove that the action of PMA on GLYT2 involves PKC (protein kinase C)-dependent and -independent pathways, although an important fraction of the effects are PKC-mediated. We show the additional participation of signalling pathways triggered by the small GTPase Rac1 on PMA action. GLYT2 inhibition by PMA and monensin also take place in brainstem primary neurons and synaptosomes, pointing to a GLYT2 trafficking regulation in the central nervous system.

The neuronal glycine transporter GLYT2 associates with membrane rafts: functional modulation by lipid environment.

The neuronal glycine transporter GLYT2 is a plasma membrane protein that removes the neurotransmitter glycine from the synaptic cleft, thereby aiding the pre-synaptic terminal reloading and the termination of the glycinergic signal. Missense mutations in the gene encoding GLYT2 (SLC6A5) cause hyperekplexia in humans. The activity of GLYT2 seems to be highly regulated. In this report, we demonstrate that GLYT2 is associated with membrane rafts in the plasma membrane of brainstem terminals and neurons. The transporter is localized to Triton X-100-insoluble light synaptosomal membranes together with flotillin-1, a marker protein for membrane rafts, in a methyl-beta-cyclodextrin (MbetaCD)-sensitive manner. In brainstem primary neurons, the GLYT2 punctuate pattern visualized by confocal microscopy was modified by cholesterol depletion with MbetaCD, unlike other non-raft neuronal markers. GLYT2-associated gold particles were observed by electron microscopy on purified rafts from brainstem synaptosomes. Furthermore, either in brainstem terminals and cultured neurons, the pharmacological reduction of the levels of raft components, cholesterol and sphingomyelin, impairs both the association of GLYT2 with membrane rafts and its transport activity. Thus, GLYT2 may require membrane raft location for optimal function, and therefore the lipid environment may constitute a new mechanism to modulate GLYT2.

Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport.

Neurotransmitter removal from glycine-mediated synapses relies on two sodium-driven high-affinity plasma membrane GlyTs that control neurotransmitter availability. Mostly glial GlyT1 is the main regulator of glycine synaptic levels, whereas neuronal GlyT2 promotes the recycling of synaptic glycine and supplies neurotransmitter for presynaptic vesicle refilling. The GlyTs differ in sodium:glycine symport stoichiometry, showing GlyT1 a 2:1 and GlyT2 a 3:1 sodium:glycine coupling. Sodium binds to the GlyTs at two conserved Na+ sites: Na1 and Na2. The location of GlyT2 Na3 site remains unknown, although Glu650 has been involved in the coordination. Here, we have used comparative MD simulations of a GlyT2 model constructed by homology to the crystalized DAT from Drosophila melanogaster by placing the Na3 ion at two different locations. By combination of in silico and experimental data obtained by biochemical and electrophysiological analysis of GlyTs mutants, we provide evidences suggesting the GlyT2 third sodium ion is held by Glu-250 and Glu-650, within a region with robust allosteric properties involved in cation-specific sensitivity. Substitution of Glu650 in GlyT2 by the corresponding methionine in GlyT1 reduced the charge-to-flux ratio to the level of GlyT1 without producing transport uncoupling. Chloride dependence of glycine transport was almost abolished in this GlyT2 mutant but simultaneous substitution of Glu250 and Glu650 by neutral amino acids rescued chloride sensitivity, suggesting that protonation/deprotonation of Glu250 substitutes chloride function. The differential behavior of equivalent GlyT1 mutations sustains a GlyT2-specific allosteric coupling between the putative Na3 site and the chloride site.

Transmembrane domains 1 and 3 of the glycine transporter GLYT1 contain structural determinants of N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-propyl]sarcosine specificity.

The neurotransmitter glycine is removed from the synaptic cleft by two Na(+)-and Cl(-)-dependent transporters: GLYT1 and GLYT2. GLYT1, expressed in glial processes of glycinergic areas and in glia and neurons of glutamatergic pathways that contain N-methyl-d-aspartate (NMDA) receptors, is essential for regulating glycine levels both at glycinergic and NMDA-containing synapses. GLYT2 is the transporter present in glycinergic neurons and provides cytosolic glycine for vesicular release from glycinergic terminals. GLYT1 is selectively inhibited by the sarcosine derivative N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-propyl]sarcosine (NFPS). In the present report, GLYT1-GLYT2 chimeric transporters have been generated and their inhibition by NFPS has been studied. The introduction of GLYT2 transmembrane domains (TMs) 1 or 3, but not 2, on GLYT1 structure reduced the inhibition potency of NFPS and sarcosine. Binding studies and kinetic analysis of NFPS inhibition indicate lower affinity and smaller sensitivity of the chimeras to the compound. Opposite chimeras containing TM1 or TM3 of GLYT1 on GLYT2 structure became sensitive to NFPS. Individual substitution mutants of GLYT2 TM1 residues on GLYT1 and opposite GLYT1 TM1 residues on GLYT2 indicate that the more N-terminal portion of GLYT1 including residue E40 contributes to NFPS specificity. Our results demonstrate that TM1 and TM3, but not TM2, contain residues involved in the specific action of NFPS on GLYT1.

Glycine transporters GlyT1 and GlyT2 are differentially modulated by glycogen synthase kinase 3ß.

Inhibitory glycinergic neurotransmission is terminated by the specific glycine transporters GlyT1 and GlyT2 which actively reuptake glycine from the synaptic cleft. GlyT1 is associated with both glycinergic and glutamatergic pathways, and is the main regulator of the glycine levels in the synapses. GlyT2 is the main supplier of glycine for vesicle refilling, a process that is vital to preserve the quantal glycine content in synaptic vesicles. Therefore, to control glycinergic neurotransmission efficiently, GlyT1 and GlyT2 activity must be regulated by diverse neuronal and glial signaling pathways. In this work, we have investigated the possible functional modulation of GlyT1 and GlyT2 by glycogen synthase kinase 3 (GSK3ß). This kinase is involved in mood stabilization, neurodegeneration and plasticity at excitatory and inhibitory synapses. The co-expression of GSK3ß with GlyT1 or GlyT2 in COS-7 cells and Xenopus laevis oocytes, leads to inhibition and stimulation of GlyT1 and GlyT2 activities, respectively, with a decrease of GlyT1, and an increase in GlyT2 levels at the plasma membrane. The specificity of these changes is supported by the antagonism exerted by a catalytically inactive form of the kinase and through inhibitors of GSK3ß such as lithium chloride and TDZD-8. GSK3ß also increases the incorporation of 32Pi into GlyT1 and decreases that of GlyT2. The pharmacological inhibition of the endogenous GSK3ß in neuron cultures of brainstem and spinal cord leads to an opposite modulation of GlyT1 and GlyT2.Our results suggest that GSK3ß is important for stabilizing and/or controlling the expression of functional GlyTs on the neural cell surface.