title: Determination of kainate receptor subunit ratios in mouse brain using novel chimeric protein standards.
abstract: Kainate-type glutamate receptors (KARs) are tetrameric channels assembled from GluK1-5. GluK1-3 are low-affinity subunits that form homomeric and heteromeric KARs, while GluK4 and GluK5 are high-affinity subunits that require co-assembly with GluK1-3 for functional expression. Although the subunit composition is thought to be highly heterogeneous in the brain, the distribution of KAR subunits at the protein level and their relative abundance in given regions of the brain remain largely unknown. In the present study, we titrated C-terminal antibodies to each KAR subunit using chimeric GluA2-GluK fusion proteins, and measured their relative abundance in the P2 and post-synaptic density (PSD) fractions of the adult mouse hippocampus and cerebellum. Analytical western blots showed that GluK2 and GluK3 were the major KAR subunits, with additional expression of GluK5 in the hippocampus and cerebellum. In both regions, GluK4 was very low and GluK1 was below the detection threshold. The relative amount of low-affinity subunits (GluK2 plus GluK3) was several times higher than that of high-affinity subunits (GluK4 plus GluK5) in both regions. Of note, the highest ratio of high-affinity subunits to low-affinity subunits was found in the hippocampal PSD fraction (0.32), suggesting that heteromeric receptors consisting of high- and low-affinity subunits highly accumulate at hippocampal synapses. In comparison, this ratio was decreased to 0.15 in the cerebellar PSD fraction, suggesting that KARs consisting of low-affinity subunits are more prevalent in the cerebellum. Therefore, low-affinity KAR subunits are predominant in the brain, with distinct subunit combinations between the hippocampus and cerebellum. Kainate receptors, an unconventional member of the iGluR receptor family, have a tetrameric structure assembled from low-affinity (GluK1-3) and high-affinity (GluK4 and GluK5) subunits. We used a simple but novel procedure to measure the relative abundance of both low- and high-affinity subunits. This method revealed that the relative amount of GluK2 plus GluK3 subunits was several times higher than that of GluK4 plus GluK5 subunits, in both the hippocampus and cerebellum.
authors.affiliation: Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan.|CREST, Japan Science and Technology Agency, Chiyoda-ku, Japan.
authors.forename: Izumi
authors.initials: I
authors.lastname: Watanabe-Iida
authors.affiliation: CREST, Japan Science and Technology Agency, Chiyoda-ku, Japan.|Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan.
authors.forename: Kohtarou
authors.initials: K
authors.lastname: Konno
authors.affiliation: Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan.|CREST, Japan Science and Technology Agency, Chiyoda-ku, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Akashi
authors.affiliation: Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan.|CREST, Japan Science and Technology Agency, Chiyoda-ku, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Abe
authors.affiliation: Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan.|CREST, Japan Science and Technology Agency, Chiyoda-ku, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Natsume
authors.affiliation: CREST, Japan Science and Technology Agency, Chiyoda-ku, Japan.|Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan.
authors.forename: Masahiko
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata, Japan.|CREST, Japan Science and Technology Agency, Chiyoda-ku, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Sakimura
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D005786:Gene Expression Regulation / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D057907:Post-Synaptic Density / Q000378:metabolism; D055503:Protein Multimerization; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D018091:Receptors, AMPA / Q000235:genetics / Q000378:metabolism; D018092:Kainic Acid Receptors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013569:Synapses; D014162:Transfection
chemical_list: D021122:Protein Subunits; D012333:RNA, Messenger; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D011993:Recombinant Fusion Proteins; C104722:glutamate receptor ionotropic, AMPA 2
keywords: GluK2; GluK3; antibody; kainate receptor; quantification; western blot