Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1.
The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.
Institut für Biochemie und Molekularbiologie, Campus Benjamin Franklin, Charité-Universitätsmedizin Berlin, Arnimallee 22, 14195 Berlin-Dahlem, Germany.
Jing
J
Hu
Jian
J
Fei
Werner
W
Reutter
Hua
H
Fan
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D050485:GABA Plasma Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D058805:GABA Uptake Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism*; D009439:Neuraminidase / Q000494:pharmacology; D010084:Oxidation-Reduction; D010504:Periodic Acid / Q000494:pharmacology; D011485:Protein Binding; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D050485:GABA Plasma Membrane Transport Proteins; D058805:GABA Uptake Inhibitors; D011993:Recombinant Fusion Proteins; D010504:Periodic Acid; D049452:Green Fluorescent Proteins; D005680:gamma-Aminobutyric Acid; D012964:Sodium; C009288:metaperiodate; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid