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

J Biol Chem. 2014 Dec 5;289(49):34308-24. doi: 10.1074/jbc.M114.586966. Epub 2014 Oct 14.

Abstract

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.

Keywords: Calcium ATPase; GlyT2; Lipid Raft; Neurotransmitter Transport; Proteomics; Sodium-Calcium Exchange.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Brain Stem / cytology
  • Brain Stem / drug effects
  • Brain Stem / metabolism
  • Gene Expression Regulation
  • Glycine Plasma Membrane Transport Proteins / genetics
  • Glycine Plasma Membrane Transport Proteins / metabolism*
  • Intercellular Signaling Peptides and Proteins
  • Male
  • Membrane Microdomains / chemistry
  • Membrane Microdomains / drug effects
  • Membrane Microdomains / metabolism
  • Peptides / pharmacology
  • Plasma Membrane Calcium-Transporting ATPases / antagonists & inhibitors
  • Plasma Membrane Calcium-Transporting ATPases / genetics
  • Plasma Membrane Calcium-Transporting ATPases / metabolism*
  • Presynaptic Terminals / drug effects
  • Primary Cell Culture
  • Protein Binding
  • Rats
  • Rats, Wistar
  • Sensory Receptor Cells / cytology
  • Sensory Receptor Cells / drug effects
  • Sensory Receptor Cells / metabolism*
  • Sodium-Calcium Exchanger / antagonists & inhibitors
  • Sodium-Calcium Exchanger / genetics
  • Sodium-Calcium Exchanger / metabolism*
  • Spinal Cord / cytology
  • Spinal Cord / drug effects
  • Spinal Cord / metabolism
  • Synaptic Transmission
  • Thiourea / analogs & derivatives
  • Thiourea / pharmacology
  • beta-Cyclodextrins / pharmacology

Substances

  • 2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate
  • Glycine Plasma Membrane Transport Proteins
  • Intercellular Signaling Peptides and Proteins
  • Peptides
  • Slc6a5 protein, rat
  • Sodium-Calcium Exchanger
  • beta-Cyclodextrins
  • caloxin 2A1
  • methyl-beta-cyclodextrin
  • sodium-calcium exchanger 1
  • Plasma Membrane Calcium-Transporting ATPases
  • Thiourea