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.
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.
Cai
Guoqiang
G
Institute of Biochemistry and Cell Biology, SIBS, CAS, Shanghai, China.
Salonikidis
Petrus S
PS
Fei
Jian
J
Schwarz
Wolfgang
W
Schülein
Ralf
R
Reutter
Werner
W
Fan
Hua
H
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050485:GABA Plasma Membrane Transport Proteins; D017930:Genes, Reporter; D006031:Glycosylation; D007700:Kinetics; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D004791:Enzyme Inhibitors; D050485:GABA Plasma Membrane Transport Proteins; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D017485:1-Deoxynojirimycin; D005680:gamma-Aminobutyric Acid; D012964:Sodium