title: Na+/K+-ATPase is a new interacting partner for the neuronal glycine transporter GlyT2 that downregulates its expression in vitro and in vivo.
abstract: 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.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain.
authors.forename: Jaime
authors.initials: J
authors.lastname: de Juan-Sanz
authors.forename: Enrique
authors.initials: E
authors.lastname: Núñez
authors.forename: Lucía
authors.initials: L
authors.lastname: Villarejo-López
authors.forename: Daniel
authors.initials: D
authors.lastname: Pérez-Hernández
authors.forename: Alejo E
authors.initials: AE
authors.lastname: Rodriguez-Fraticelli
authors.forename: Beatriz
authors.initials: B
authors.lastname: López-Corcuera
authors.forename: Jesús
authors.initials: J
authors.lastname: Vázquez
authors.forename: Carmen
authors.initials: C
authors.lastname: Aragón
mesh_terms: D000818:Animals; D001933:Brain Stem / Q000166:cytology; D015536:Down-Regulation; D004705:Endocytosis; D018507:Gene Expression Regulation, Developmental; D050487:Glycine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D006706:Homeostasis; D008297:Male; D021962:Membrane Microdomains / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D050487:Glycine Plasma Membrane Transport Proteins; C507810:SLC6A5 protein, zebrafish; C493269:Slc6a5 protein, rat; D029961:Zebrafish Proteins; D010042:Ouabain; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase