[3H]kainate localization in goldfish brain: receptor autoradiography and membrane binding.

The anatomical distribution of specific [3H]kainate binding in goldfish brain was investigated by membrane binding and autoradiographical techniques. Saturation binding of the radioligand was determined in 8 anatomically defined regions and demonstrated a single class of high affinity sites with Kd values ranging from 290 to 650 nM. Kainate receptor densities, however, varied significantly. The cerebellum contained the highest concentration of binding sites (964 pmol/mg prot.), while the optic tectum had the lowest (96 pmol/mg prot.). Binding site distributions determined by autoradiographic studies demonstrated the same regional variation and allowed more specific localization of the binding sites. Within the cerebellum, the molecular layers of the corpus, valvula and lobus caudalis displayed a uniform and highly intense image while the granule cell layers (except for the medial granule cell mass of the lobus caudalis) did not. Other areas of intense binding were the posterior tubercle of the diencephalon, inferior lobes of the hypothalamus and layers 1 and 2 of the optic tectum (deep to the periventricular granule cells).

Coupling of a purified goldfish brain kainate receptor with a pertussis toxin-sensitive G protein.

Goldfish brain has a high density of [3H]kainate-binding sites, a subpopulation of which appears to be coupled to a pertussis toxin-sensitive G protein. We show here that a purified kainate receptor preparation reconstituted into phospholipid vesicles exhibits guanine nucleotide-sensitive high-affinity [3H]kainate binding. Pertussis toxin treatment abolishes the guanine nucleotide-sensitive portion of the [3H]kainate binding, and kainate promotes [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding and [gamma-32P]GTP hydrolysis. Guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) decreases the apparent Stokes radius of the soluble purified receptor preparation, consistent with dissociation of the kainate receptor-G protein complexes. The affinity-purified preparations contain proteins of 45, 41, and 35 kDa. The 45- and 41-kDa proteins crossreact with antibodies against the kainate receptor cloned from frog brain. The 35-kDa protein is recognized by an antiserum (SW) directed against the beta subunit of G proteins. When kainate receptors are purified in the presence of GTP[gamma S], the 35-kDa protein is no longer present. Also, [3H]kainate affinity is decreased and is no longer guanine nucleotide sensitive. Upon reconstitution with purified G proteins, high-affinity guanine nucleotide-sensitive binding and kainate-stimulated GTPase activity can be restored. These observations indicate that a kainate receptor from goldfish brain functionally interacts with a pertussis toxin-sensitive G protein.

Biochemical characterization of kainate receptors from goldfish brain.

Kainate receptors from goldfish brain were purified by affinity chromatography. Unlike previously published purifications, which have yielded single proteins of 48-50 kDa from frog, chick, and pigeon brain, our preparations contained two polypeptides, of 41 kDa and 45 kDa. In addition, a broad band centered at 120 kDa was present. Some of the 41-kDa and 45-kDa polypeptides were derived from the higher molecular mass protein. All of these proteins were recognized by a monoclonal antibody produced against a purified frog kainate receptor. The distribution of the 41-kDa and 45-kDa proteins varied independently in different major brain regions, suggesting that they can exist as separate independent proteins. A partial amino acid sequence of the 41-kDa polypeptide is very similar (40-60% identity) to specific segments of the frog and chick kainate-binding proteins and the alpha-amino-3-hydroxy-5-methylisoxazolepropionate/kainate ion channels. The characteristics of the 41-kDa and 45-kDa polypeptides suggest that these two proteins are distinct. Photo-affinity labeling with [3H]kainate showed that a [3H]kainate binding site is associated with the 41-kDa polypeptide, and peptide mapping suggests that the two proteins are not identical. In addition, the two peptides do not appear to be related by differential glycosylation or phosphorylation. The 41-kDa and 45-kDa polypeptides, therefore, appear to be distinct and may represent kainate receptor subtypes or, in some cases, possibly two different subunits of a kainate receptor complex.

The interaction of a kainate receptor from goldfish brain with a pertussis toxin-sensitive GTP-binding protein.

Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.