Isolation, localization, and cloning of a kainic acid binding protein from frog brain.
Excitatory amino acids (EAA) are major neurotransmitters in the vertebrate central nervous system. EAA receptors have been divided into three major subtypes on the basis of electrophysiological and ligand binding studies: N-methyl-D-aspartate, kainate, and quisqualate receptors. To understand their molecular properties, we undertook a project aimed at isolation and cloning of these receptor subtypes. We purified a kainate binding protein (KBP) from frog brain, in which kainate binding sites are about fortyfold more abundant than in rat brain, using domoic acid affinity chromatography, and made monoclonal and polyclonal antibodies to the purified protein. These antibodies immunoprecipitate the frog KBP but not KBPs from other species. Immunocytochemical analyses show that KBP has a synaptic and extrasynaptic localization in frog optic tectum, with most labeling being extrasynaptic. The cDNA encoding frog brain KBP was isolated by screening a frog brain cDNA library with oligonucleotide probes that were based on the amino acid sequence of the purified protein. The deduced amino acid sequence of the KBP has a hydrophobic profile similar to those of other ligand-gated ion channel subunits, such as the nicotinic acetylcholine receptor, the GABAA receptor, and the glycine receptor. Frog brain KBP is very similar (36% amino acid identity to the carboxyl half) to rat brain kainate receptor, suggesting that these two proteins evolved from a common ancestor. The function of KBP in frog brain remains a major question. Preliminary results showed that Xenopus laevis oocytes injected with KBP RNA did not produce a detectable electrophysiological response when perfused with kainate. These results suggest that additional subunits may be required to form a functional receptor or that KBP is not functionally related to a neurotransmitter receptor.
Wenthold
R J
RJ
Section of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892.
Hampson
D R
DR
Wada
K
K
Hunter
C
C
Oberdorfer
M D
MD
Dechesne
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001001:Anura; D001923:Brain Chemistry; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter / Q000032:analysis / Q000737:chemistry / Q000235:genetics
D004247:DNA; D018092:Kainic Acid Receptors; D017981:Receptors, Neurotransmitter