The role of N-glycosylation in the stability, trafficking and GABA-uptake of GABA-transporter 1. Terminal N-glycans facilitate efficient GABA-uptake activity of the GABA transporter

FEBS J. 2005 Apr;272(7):1625-38. doi: 10.1111/j.1742-4658.2005.04595.x.

Abstract

Neurotransmitter transporters play a major role in achieving low concentrations of their respective transmitter in the synaptic cleft. The GABA transporter GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins which possess 12 putative transmembrane domains and three N-glycosylation sites in the extracellular loop between transmembrane domain 3 and 4. To study the significance of N-glycosylation, green fluorescence protein (GFP)-tagged wild type GAT1 (NNN) and N-glycosylation defective mutants (DDQ, DGN, DDN and DDG) were expressed in CHO cells. Compared with the wild type, all N-glycosylation mutants showed strongly reduced protein stability and trafficking to the plasma membrane, which however were not affected by 1-deoxymannojirimycin (dMM). This indicates that N-glycosylation, but not terminal trimming of the N-glycans is involved in the attainment of a correctly folded and stable conformation of GAT1. All N-glycosylation mutants were expressed on the plasma membrane, but they displayed markedly reduced GABA-uptake activity. Also, inhibition of oligosaccharide processing by dMM led to reduction of this activity. Further experiments showed that both N-glycosylation mutations and dMM reduced the V(max) value, while not increasing the K(m) value for GABA uptake. Electrical measurements revealed that the reduced transport activity can be partially attributed to a reduced apparent affinity for extracellular Na+ and slowed kinetics of the transport cycle. This indicates that N-glycans, in particular their terminal trimming, are important for the GABA-uptake activity of GAT1. They play a regulatory role in the GABA translocation by affecting the affinity and the reaction steps associated with the sodium ion binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Deoxynojirimycin / pharmacology
  • Animals
  • CHO Cells
  • Cell Membrane / metabolism
  • Cricetinae
  • Cricetulus
  • Enzyme Inhibitors / pharmacology
  • GABA Plasma Membrane Transport Proteins
  • Genes, Reporter
  • Glycosylation
  • Kinetics
  • Membrane Transport Modulators
  • Membrane Transport Proteins / antagonists & inhibitors
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Polysaccharides / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sodium / metabolism
  • gamma-Aminobutyric Acid / metabolism*

Substances

  • Enzyme Inhibitors
  • GABA Plasma Membrane Transport Proteins
  • Membrane Transport Modulators
  • Membrane Transport Proteins
  • Polysaccharides
  • Recombinant Fusion Proteins
  • 1-Deoxynojirimycin
  • gamma-Aminobutyric Acid
  • Sodium