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

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 discoveryThis work was supported by grants of the Spanish ‘Ministerio de Economía y Competitividad’, grant number SAF2017-84235-R (AEI/FEDER, EU) to B.L.-C. and by institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMS

Regulación de la neurotransmisión glicinérgica en procesos de dolor inflamatorio: una nueva vía de acción de la prostaglandina E2 en médula espinal

Glycinergic inhibitory neurotransmission is terminated by reuptake through specific transporters, GlyT1 (glial isoform) and GlyT2 (neuronal isoform). In this work we describe that Prostaglandin E2 (PGE2, an important mediator of inflammatory pain) activates GlyT2-mediated recapture of glycine via interaction with the EP3 receptor. Moreover, in these conditions a diminished ubiquitination of GlyT2 is observed. Ubiquitination is an important modification for the correct trafficking of this transporter.  We propose that the reduction of ubiquitination leads to accumulate GlyT2 in the neuronal surface, which could explain the PGE2-mediated activation of GlyT2. Therefore, our results suggest that GlyT2 is an interesting therapeutic target and its inhibition could contribute to reduce inflammatory pain.La acción de la glicina como neurotransmisor inhibidor es finalizada por su recaptación del espacio sináptico a través de dos transportadores específicos, GlyT1 (isoforma glial) y GlyT2 (isoforma neuronal). En este trabajo describimos un mecanismo mediante el cual la unión de la prostaglandina E2 (un importante mediador del dolor inflamatorio) a sus receptores EP3 activa la recaptación de glicina llevada a cabo por GlyT2. Esta activación coincide con una disminución de la ubiquitinación del transportador, modificación post-traduccional necesaria para su correcto tráfico intracelular. Una menor ubiquitinación de GlyT2 produce una acumulación del transportador en la superficie neuronal, lo que explica la activación observada. Por tanto, los resultados de este trabajo sugieren que GlyT2 es una interesante diana terapéutica cuya inhibición podría contribuir a la reducción del dolor inflamatorio

ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis

ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering

padA: un nuevo gen implicado en diferenciación y desarrollo en Dictyostelium discoideum

Leída en Universidad Complutense. Facultad de Ciencias Biológicas el 01/18/2008; 136 págs.Dictyostelium es una ameba que vive en el suelo alimentándose de bacterias. Las señales de ausencia de nutrientes, desencadenan un complejo programa morfogenético que conlleva la formación de un cuerpo fructífero con dos tipos celulares perfectamente diferenciados, esporas y células tallo. La diferenciación de estos dos tipos celulares requiere una compleja regulación de rutas de señalización en las que diferentes señales (AMP cíclico, DIF-1, amonio) juegan un papel clave en la especificación de un tipo celular u otro. El AMPc, es esencial para el inicio del proceso de diferenciación y desarrollo. El morfógeno DIF-1, induce la diferenciación a células tallo y reprime la formación de esporas. El amonio, se genera como consecuencia de la intensa actividad metabólica y actúa como una base débil inhibiendo la diferenciación de ambos tipos celulares y regulando el inicio de la culminación (Stremcki et al., 2005; Williams, 2006). Este trabajo de tesis doctoral se ha basado en la caracterización genética y molecular de un mutante deficiente en la diferenciación de células tallo que hemos llamado, padA. El mutante fue aislado a partir de una genoteca, construída mediante la técnica REMI (Kuspa y Loomis, 1992), por su incapacidad para inducir la expresión del gen pretallo ecmB en presencia de DIF-1 (Sarafimidis, 2003). El mutante termosensible padA, recapitulado en el fondo silvestre, presenta una disrupción terminal en el gen padA que codifica una proteína truncada en la región carboxi-terminal. La proteína mantiene actividad residual a la temperatura óptima de crecimiento, 22ºC y carece de actividad a la temperatura restrictiva, 27ºC. La termosensibilidad del alelo padA nos permite estudiar las funciones de un gen esencial para el crecimiento y desarrollo de D. discoideum. El fenotipo del mutante padA es pleitrópico, a 22ºC, el crecimiento vegetativo es más lento y el desarrollo presenta un retraso general. A 27ºC, las amebas mutantes no son capaces de crecer en medio axénico, mientras que el tipo silvestre lo hace con normalidad hasta 30ºC (Saito et al., 2005). A esta temperatura, el mutante presenta un bloqueo en el desarrollo y las pocas estructuras que consiguen superarlo, forman unas estructuras aberrantes que mantienen la verticalidad y recuersan a un cuerpo fructífero, carecen de células tallo y esporas perfectamente diferenciadas. El análisis estructural in silico de la proteína PadA, así como las mutaciones puntuales realizadas en los dominios funcionales de la proteína, nos ha permitido demostrar que PadA es un miembro de la superfamilia estructural de proteínas "NAD/P-Rossman fold" y pertenece a la familia de las hidrógeno reductasas de cadena corta o "SDR-proteíns". Dentro de esta familia los homólogos más próximos a PadA se encuentran en el grupo de los reguladores transcripcionales negativos, representado por las proteínas Nmr1 y Nmr2, identificadas y aisladas en Neurospora crassa y Aspergillus nidulans, respectivamente (Fu y Marzluf, 1988; Andrianopaulos et al., 1998). Tanto Nmr1 como Nmr2, son co-represores transcripcionales en la ruta de represión por metabolito de amonio, que regula el patrón de expresión de genes del metabolismo primario y secundario en hongos (Wilson y Arst, 1998). La actividad de estas proteínas está modulada por la unión con NAD/NADP, que regula la interacción con los factores de transcripción tipo GATA (Stammers, 2001; Lamb et al., 2004). En Dictyostelium, es la primera vez que se describen este tipo de proteínas NmrA y el análisis del genoma predice que existen 21 factores de transcripción tipo GATA (Eichiger et al., 2004). El descubrimiento de este nuevo tipo de reguladores transcripcionales en Dictyostelium pone de manifiesto la elevada complejidad de los mecanismos de regulación que controlan la diferenciación celular en este organismo.Peer reviewe

Constitutive Endocytosis and Turnover of the Neuronal Glycine Transporter GlyT2 Is Dependent on Ubiquitination of a C-Terminal Lysine Cluster

Inhibitory glycinergic neurotransmission is terminated by sodium and chloride-dependent plasma membrane glycine transporters (GlyTs). The mainly glial glycine transporter GlyT1 is primarily responsible for the completion of inhibitory neurotransmission and the neuronal glycine transporter GlyT2 mediates the reuptake of the neurotransmitter that is used to refill synaptic vesicles in the terminal, a fundamental role in the physiology and pathology of glycinergic neurotransmission. Indeed, inhibitory glycinergic neurotransmission is modulated by the exocytosis and endocytosis of GlyT2. We previously reported that constitutive and Protein Kinase C (PKC)-regulated endocytosis of GlyT2 is mediated by clathrin and that PKC accelerates GlyT2 endocytosis by increasing its ubiquitination. However, the role of ubiquitination in the constitutive endocytosis and turnover of this protein remains unexplored. Here, we show that ubiquitination of a C-terminus four lysine cluster of GlyT2 is required for constitutive endocytosis, sorting into the slow recycling pathway and turnover of the transporter. Ubiquitination negatively modulates the turnover of GlyT2, such that increased ubiquitination driven by PKC activation accelerates transporter degradation rate shortening its half-life while decreased ubiquitination increases transporter stability. Finally, ubiquitination of GlyT2 in neurons is highly responsive to the free pool of ubiquitin, suggesting that the deubiquitinating enzyme (DUB) ubiquitin C-terminal hydrolase-L1 (UCHL1), as the major regulator of neuronal ubiquitin homeostasis, indirectly modulates the turnover of GlyT2. Our results contribute to the elucidation of the mechanisms underlying the dynamic trafficking of this important neuronal protein which has pathological relevance since mutations in the GlyT2 gene (SLC6A5) are the second most common cause of human hyperekplexia. © 2013 de Juan-Sanz et al.Spanish Direccion General de Investigacion Cientıfica y Tecnica (SAF2008-05436; SAF2011-28674); Fondo de Investigaciones Sanitarias (CIBERER); Comunidad Autonoma de Madrid; Fundacion Ramon ArecesPeer Reviewe

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 proposeSpanish ‘Ministerio de Economia y Competitividad’, grant number SAF2017-84235-R (AEI/FEDER, EU) to B.L.-C. and by institutional grants from the Fundacion Ramon Areces and Banco de Santande

A new protein carrying an NmrA-like domain is required for cell differentiation and development in Dictyostelium discoideum

12 páginas, 10 figuras, 1 tabla, 3 figuras suplementarias -- PAGS nros. 331-342We have isolated a Dictyostelium mutant unable to induce expression of the prestalk-specific marker ecmB in monolayer assays. The disrupted gene, padA, leads to a range of phenotypic defects in growth and development. We show that padA is essential for growth, and we have generated a thermosensitive mutant allele, padA−. At the permissive temperature, mutant cells grow poorly; they remain longer at the slug stage during development and are defective in terminal differentiation. At the restrictive temperature, growth is completely blocked, while development is permanently arrested prior to culmination. padA− slugs are deficient in prestalk A cell differentiation and present an abnormal ecmB expression pattern. Sequence comparisons and predicted three-dimensional structure analyses show that PadA carries an NmrA-like domain. NmrA is a negative transcriptional regulator involved in nitrogen metabolite repression in Aspergillus nidulans. PadA predicted structure shows a NAD(P)+-binding domain, which we demonstrate that is essential for function. We show that padA− development is more sensitive to ammonia than wild-type cells and two ammonium transporters, amtA and amtC, appear derepressed during padA− development. Our data suggest that PadA belongs to a new family of NAD(P)+-binding proteins that link metabolic changes to gene expression and is required for growth and normal developmenThis work was supported by a grant from the Spanish Ministerio de Educación y Ciencia, Dirección General de Investigación (BFU2006-02295)Peer reviewe

UCHL1 inhibition impairs Glyt2 constitutive endocytosis in neurons.

<p>A) Representative immunoblot of brainstem and spinal cord primary neurons. Cells were pretreated for 2 h with vehicle (DMSO) or <i>LDN</i>-57444 (UCHL1 inhibitor: 10 μM) and were then exposed to monensin (35 μM, 30 min) or the vehicle alone (EtOH), in the presence or absence of UCHL1. Cell surface proteins were labeled with sulfo-NHS-SS-biotin and the biotinylated proteins were pulled down with streptavidin-agarose beads. GlyT2 expression was analyzed in Western blots using calnexin immunodetection as a control of intracellular non-biotinylated protein. B, biotinylated protein (30 μg); T, total protein (10 μg). B) Densitometric analysis of four independent Western blots as in (A) relative to the control values (Veh). Data represent the means ± SEM. **, significant difference with respect to control; p<0.01 (ANOVA with Tukey's post-hoc test). C) Primary neurons were incubated with vehicle or <i>LDN</i>-57444 as described above, the cells were lysed and the ubiquitinated GlyT2 was immunoprecipitated with agarose-conjugated anti-multiubiquitin antibody. The immunoprecipitates were probed with anti-GlyT2 antibody. Ub, anti-multiubiquitin immunoprecipitation (75 μg); T, total protein (10 μg). D) Quantification of four experiments performed as described in (C). Bars represent the mean ± SEM level of <i>LDN</i>-57444-treated ubiquitinated transporter relative to that of ubiquitinated transporter in vehicle-treated cells. *, significant difference with respect to control; p<0.05 (Student's t-test).</p