title: Molecular basis of the dominant negative effect of a glycine transporter 2 mutation associated with hyperekplexia.
abstract: Hyperekplexia or startle disease is a rare clinical syndrome characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. This neurological disorder can have serious consequences in neonates, provoking brain damage and/or sudden death due to apnea episodes and cardiorespiratory failure. Hyperekplexia is caused by defective inhibitory glycinergic neurotransmission. Mutations in the human SLC6A5 gene encoding the neuronal GlyT2 glycine transporter are responsible for the presynaptic form of the disease. GlyT2 mediates synaptic glycine recycling, which constitutes the main source of releasable transmitter at glycinergic synapses. Although the majority of GlyT2 mutations detected so far are recessive, a dominant negative mutant that affects GlyT2 trafficking does exist. In this study, we explore the properties and structural alterations of the S512R mutation in GlyT2. We analyze its dominant negative effect that retains wild-type GlyT2 in the endoplasmic reticulum (ER), preventing surface expression. We show that the presence of an arginine rather than serine 512 provoked transporter misfolding, enhanced association to the ER-chaperone calnexin, altered association with the coat-protein complex II component Sec24D, and thereby impeded ER exit. The S512R mutant formed oligomers with wild-type GlyT2 causing its retention in the ER. Overexpression of calnexin rescued wild-type GlyT2 from the dominant negative effect of the mutant, increasing the amount of transporter that reached the plasma membrane and dampening the interaction between the wild-type and mutant GlyT2. The ability of chemical chaperones to overcome the dominant negative effect of the disease mutation on the wild-type transporter was demonstrated in heterologous cells and primary neurons.
authors.affiliation: From the Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid 28049, Spain, the IdiPAZ-Hospital Universitario La Paz, Universidad Autónoma de Madrid, Madrid 28046, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Arribas-González
authors.affiliation: From the Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid 28049, Spain, the IdiPAZ-Hospital Universitario La Paz, Universidad Autónoma de Madrid, Madrid 28046, Spain the Centro de Investigación Biomédica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Madrid 28029, Spain, and.
authors.forename: Jaime
authors.initials: J
authors.lastname: de Juan-Sanz
authors.affiliation: From the Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid 28049, Spain, the IdiPAZ-Hospital Universitario La Paz, Universidad Autónoma de Madrid, Madrid 28046, Spain the Centro de Investigación Biomédica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Madrid 28029, Spain, and.
authors.forename: Carmen
authors.initials: C
authors.lastname: Aragón
authors.affiliation: From the Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid 28049, Spain, the IdiPAZ-Hospital Universitario La Paz, Universidad Autónoma de Madrid, Madrid 28046, Spain the Centro de Investigación Biomédica en Red de Enfermedades Raras, Instituto de Salud Carlos III, Madrid 28029, Spain, and blopez@cbm.csic.es.
authors.forename: Beatriz
authors.initials: B
authors.lastname: López-Corcuera
mesh_terms: D000818:Animals; D019921:Biotinylation; D019556:COS Cells; D037281:Calnexin / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D002522:Chlorocebus aethiops; D003720:Densitometry; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005799:Genes, Dominant; D005998:Glycine / Q000737:chemistry; D050487:Glycine Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D016750:Stiff-Person Syndrome / Q000235:genetics* / Q000378:metabolism; D009435:Synaptic Transmission
chemical_list: D050487:Glycine Plasma Membrane Transport Proteins; D018832:Molecular Chaperones; C493271:SLC6A5 protein, human; D037281:Calnexin; D005998:Glycine
keywords: Calnexin; Dominant Negative; Endoplasmic Reticulum (ER); Glycine; Hyperekplexia; Molecular Chaperone; Neurotransmitter Transport; Protein Structure; Protein Synthesis; Ubiquitylation (Ubiquitination)