Dominant negative mutant of ionotropic glutamate receptor subunit GluR3: implications for the role of a cysteine residue for its channel activity and pharmacological properties.
We reported that a 33-amino-acid deletion (from tyrosine-715 to glycine-747) in a putative extracellular loop of GluR3 produced a mutant that exhibited dominant negative effects upon the functional expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [Sekiguchi et al. (1994) J. Biol. Chem. 269, 14559-14565]. In this study, we searched for a key residue in the dominant negative effects to explore the mechanism and examined the role of the residue in the function of the AMPA receptor. We prepared 20 GluR3 mutants with amino acid substitutions within the 33-amino-acid-region, and dominant negative effects were tested electrophysiologically in Xenopus oocytes co-expressing the mutant and normal subunits. Among the mutants, only a GluR3 mutant in which an original cysteine (Cys)-722 was replaced by alanine exhibited a dominant negative effect comparable with that of the original mutant in which the entire 33-amino-acid segment is deleted. The co-expression of the Cys-722 mutant did not inhibit the translation of normal subunits in oocytes. The Cys-722 mutant formed a functional homomeric receptor with significantly higher affinity for glutamate or kainate than a homomeric GluR3 receptor. The Cys-722 mutation greatly enhanced the sensitivity of GluR3 for aniracetam, which alters kinetic properties of AMPA receptors. The kainate-induced currents in oocytes expressing the Cys-722 mutant alone showed strong inward rectification. These results suggest that the Cys-722 in GluR3 is important for dominant negative effects and plays a crucial role in the determination of pharmacological properties in AMPA receptor function.
Watase
K
K
Department of Degenerative Neurological Diseases, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan.
Sekiguchi
M
M
Matsui
T A
TA
Tagawa
Y
Y
Wada
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D002462:Cell Membrane; D003545:Cysteine; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D018698:Glutamic Acid / Q000494:pharmacology; D007473:Ion Channels / Q000502:physiology*; D007608:Kainic Acid / Q000494:pharmacology; D008969:Molecular Sequence Data; D009865:Oocytes; D010641:Phenotype; D017354:Point Mutation; D011487:Protein Conformation; D018091:Receptors, AMPA / Q000502:physiology*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014981:Xenopus
D007473:Ion Channels; D018091:Receptors, AMPA; D011994:Recombinant Proteins; D018698:Glutamic Acid; D003545:Cysteine; D007608:Kainic Acid