Non-essential roles of cysteine residues in functional expression and redox regulatory pathways for canine glutamate/aspartate transporter based on mutagenic analysis.
A redox regulatory mechanism and a molecular link between oxidative and excitotoxic neurodegeneration have been postulated for high-affinity Na(+)-dependent glutamate transporters. In the present study, mutations were introduced at three cysteine residues in canine glutamate/aspartate transporter (GLAST) to investigate the functional significance of thiol groups in response to oxidation. Cys(-) GLAST, in which all cysteines were replaced by other amino acids, as well as other mutants with disruption of one of three cysteine residues, showed insoluble oligomer formation, which was considered to be due to spontaneous and excessive oxidation as observed in wild-type GLAST. The mutant transporters also showed plasma-membrane localization and glutamate-transport kinetics that were very similar to those of wild-type GLAST. Glutamate-transport activities in COS-7 cells transfected with wild-type and Cys(-) GLAST were inhibited to the same degree when cells were exposed to Hg(2+) and were recovered by the addition of thiol-specific reductant dithiothreitol. These findings suggest that cysteine residues are not critical in functional expression of GLAST and the redox-sensing pathway via glutamate transporters.
Tamahara
Satoshi
S
Laboratory of Veterinary Clinical Pathobiology, Department of Veterinary Medical Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan.
Inaba
Mutsumi
M
Sato
Kota
K
Matsuki
Naoaki
N
Hikasa
Yoshiaki
Y
Ono
Ken-Ichiro
K
D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry / Q000502:physiology*; D018076:DNA, Complementary / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004285:Dogs; D018698:Glutamic Acid / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008628:Mercury / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D013687:Telencephalon / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D027322:Amino Acid Transport System X-AG; D018076:DNA, Complementary; D018698:Glutamic Acid; D012964:Sodium; D008628:Mercury; D003545:Cysteine; D010100:Oxygen; D004229:Dithiothreitol