No abstract

J. Neurochem. (2011) 118, 195–204. Abstract Glycine synaptic levels are controlled by glycine transporters (GLYTs) catalyzing Na+/Cl−/glycine cotransport. GLYT1 displays a 2 : 1 : 1 stoichiometry and is the main regulator of extracellular glycine concentrations. The neuronal GLYT2, with higher sodium coupling (3 : 1 : 1), supplies glycine to the pre‐synaptic terminal to refill synaptic vesicles. In this work, using structural homology modelling and molecular dynamics simulations of GLYTs, we predict the conservation of the two sodium sites present in the template (leucine transporter from Aquifex aeolicus), and confirm its use by mutagenesis and functional analysis. GLYTs Na1 and Na2 sites show differential cation selectivity, as inferred from the action of lithium, a non‐transport‐supporting ion, on Na+‐site mutants. GLYTs lithium responses were unchanged in Na1‐site mutants, but abolished or inverted in mutants of Na2 site, which binds lithium in the presence of low sodium concentrations and therefore, controls lithium responses. Here, we report, for the first time, that lithium exerts opposite actions on GLYTs isoforms. Glycine transport by GLYT1 is inhibited by lithium whereas GLYT2 transport is stimulated, and this effect is more evident at increased glycine concentrations. In contrast to GLYT1, high and low affinity lithium‐binding processes were detected in GLYT2.

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.

Corresponding author: Beatriz López-Corcuera, blopez@cbm.uam.es † These authors share last authorship.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.

No abstract

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.

PDK3 negatively regulates the pyruvate dehydrogenase complex (PDC) activity in the mitochondria by reversible phosphorylation of its first catalytic component (E1). PDK3 hence has a fundamental role in linking glycolysis to the energy producing Krebs cycle. The discovery of PDK3 has added to the growing list of CMT genes related to mitochondrial biology 5 , suggesting mitochondrial pathway deficits may be a common theme in some CMT neuropathies. Our previous investigations showed the p.R158H mutation produces a PDK3 enzyme with increased kinase activity 3. Experiments using CMTX6 patient fibroblasts demonstrated mutant PDK3 hyperactivity leads to increased phosphorylation of the PDC E1 subunit at specific serine residues and hence attenuation of the pyruvate dehydrogenase activity. Consequently, CMTX6 patient fibroblasts show increased lactate, decreased ATP and alteration of the mitochondrial network. Importantly, E1 hyperphosphorylation was reversed by treating the patient fibroblasts with a pan PDK inhibitor, dichloroacetic acid (DCA), opening a venue for therapeutic intervention for CMTX6 6. Despite the active research for therapies that can stop or ameliorate degeneration of axons, there is yet no cure for CMT. This fact can be explained in part by a reliance on animal models, transformed cell lines and heterologous recombinant systems for drug discovery 7. The use of human induced pluripotent stem cell (iPSC) technology has recently opened up the possibility to produce disease-relevant human models for drug discovery for inherited diseases in general and neurodegenerative disorders in particular 8. iPSC lines from CMT patients have increasingly been generated 9-11 and key pathological features for the disease have been replicated in some instances 12-15 in CMT patient derived motor neurons. In this study we have used patient fibroblasts from a recently identified family carrying the p.R158H PDK3 mutation 4 and, following confirmation of the E1 hyperphosphorylation as a CMTX6 disease signature, generated iPSCs from this patient. To eliminate the influence of variable genetic backgrounds from genetically unrelated controls, we also generated an isogenic wild type iPSC line by targeted gene correction using the CRISPR/Cas9 system. Our results show the E1 hyperphosphorylation is maintained in the CMTX6-derived iPSCs following reprograming of the patient fibroblasts and is also observed after differentiation into spinal cord motor neurons. Our data reveals abnormalities in the bioenergetic profile and mitochondrial morphological features in the CMTX6-derived motor neurons. Additionally, analyses of the organelle trafficking demonstrated the PDK3 mutation specifically affects mitochondrial trafficking in the patient motor neurons. Importantly, we have reversed the CMTX6 cellular phenotype both pharmacologically, using a pan PDK inhibitor, and by genetically correcting the p.R158H PDK3 mutation. Material and Methods Research guidelines and regulations. All research and cell culture procedures were conducted follow...

A rare form of X-linked Charcot-Marie-Tooth neuropathy, CMTX3, is caused by an interchromosomal insertion occurring at chromosome Xq27.1. Interestingly, eight other disease phenotypes have been associated with insertions (or insertion-deletions) occurring at the same genetic locus. To date, the pathogenic mechanism underlying most of these diseases remains unsolved, although local gene dysregulation has clearly been implicated in at least two phenotypes. The challenges of accessing disease-relevant tissue and modelling these complex genomic rearrangements has led to this research impasse. We argue that recent technological advancements can overcome many of these challenges, particularly induced pluripotent stem cells (iPSC) and their capacity to provide access to patient-derived disease-relevant tissue. However, to date these valuable tools have not been utilized to investigate the disease-associated insertions at chromosome Xq27.1. Therefore, using CMTX3 as a reference disease, we propose an experimental approach that can be used to explore these complex mutations, as well as similar structural variants located elsewhere in the genome. The mutational hotspot at Xq27.1 is a valuable disease paradigm with the potential to improve our understanding of the pathogenic consequences of complex structural variation, and more broadly, refine our knowledge of the multifaceted process of long-range gene regulation. Intergenic structural variation is a critically understudied class of mutation, although it is likely to contribute significantly to unsolved genetic disease.