Identification and localization of a kainate binding protein in the frog inner ear by electron microscopy immunocytochemistry.

A kainate binding protein (KBP) was studied in Rana pipiens inner ear using monoclonal and polyclonal antibodies against affinity purified KBP from frog brain. The KBP identified and analyzed in inner ear tissue homogenates, with one- and two-dimensional immunoblots, was similar to the affinity purified KBP and to the antibody-identified frog brain KBP. As brain KBP, inner ear KBP had 5 main components in the molecular weight dimension, centered at Mr = 48,000; however, inner ear KBP had a greater abundance of the higher molecular weight components. Light and electron microscopy observations showed KBP immunostaining at two locations: (1) in the dendrites of the eight nerve afferent fibers contacting sensory hair cells, with the postsynaptic density being more intensely stained; and (2) on the cytoplasmic membrane of fibroblasts present in the inner ear connective tissue which displayed intense immunostaining. The presence of kainate (KA) binding sites in the inner ear was assessed using in vitro receptor autoradiography. [3H]KA binding sites were found in connective tissue areas confirming the immunocytochemistry results. The postsynaptic localization of the KBP in afferent endings, strongly supports it as being a component of the KA receptor complex. However, its presence on fibroblasts situated in the inner ear connective tissue makes its function hypothetical. The dual presence of the KBP on non-neuronal cells as well as at postsynaptic membrane sites suggests the existence of a family of proteins involved in KA binding and KA receptors with a complex organization.

Distribution of a putative kainic acid receptor in the frog central nervous system determined with monoclonal and polyclonal antibodies: evidence for synaptic and extrasynaptic localization.

A frog brain kainic acid receptor (KAR) was studied using monoclonal and polyclonal antibodies against the affinity-purified receptor. Immunocytochemistry was done on sections of the frog CNS, and the distribution of immunostaining was compared with the distribution of high- and low-affinity 3H-kainic acid (3H-KA) binding sites determined with in vitro receptor autoradiography. These studies showed (1) similar distributions of high- and low-affinity 3H-KA binding sites, (2) identical patterns of immunostaining with the polyclonal antibodies and 2 monoclonal antibodies, and (3) an antibody binding distribution which closely matched that of 3H-KA binding, suggesting that the antibodies recognize the primary KAR in frog brain. In the frog brain, an anteroposterior gradient of immunostaining was observed, with the telencephalon intensely and uniformly immunoreactive. Other areas intensely immunoreactive included the cerebellum, the infundibulum, the tectal and posterior commissures, and the laminar nucleus of the torus semicircularis. The optic tectum showed selective staining of the plexiform layers 3 and 5-7. The pattern of staining was punctate and appeared to be associated with nerve fibers, among them dendritic arborizations. Electron microscopic observations showed staining at the cytoplasmic side of postsynaptic membranes. Extra-synaptic staining was observed as patches on the surface of unmyelinated nerve processes.

Solubilization and partial purification of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites from rat brain.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding sites were solubilized from rat brain membranes using 1% Triton X-100 in 0.5 M potassium phosphate buffer containing 20% glycerol. The solubilized binding sites were stable, permitting biochemical and pharmacological characterization as well as partial purification. Pharmacological and binding analyses indicated that the solubilized binding sites were similar to the membrane-bound sites. Both the solubilized and the membrane-bound preparations contained high- and low-affinity AMPA binding sites in the presence of potassium thiocyanate. A similar rank order for inhibition of [3H]AMPA binding by several excitatory amino acid analogs was obtained for the soluble and membrane-bound preparations. [3H]AMPA binding to both soluble and membrane-bound preparations was increased in the presence of potassium thiocyanate. The solubilized AMPA binding sites migrated as a single peak with gel filtration chromatography, with an Mr of 425,000. Beginning with the solubilized preparation, AMPA binding sites were purified 54-fold with ion-exchange chromatography and gel filtration. The characterization and purification of these soluble binding sites is potentially useful for the molecular characterization of this putative excitatory amino acid receptor subtype.

Identification and characterization of the ligand binding subunit of a kainic acid receptor using monoclonal antibodies and peptide mapping.

Monoclonal antibodies (mAb) and a polyclonal antiserum were produced against a kainic acid receptor (KAR) purified from frog brain. Several of the mAb and the antiserum immunoprecipitated [3H]kainic acid binding activity from solubilized preparations of frog brain and labeled a group of proteins on immunoblots that migrated at Mr = 48,000. These results confirm that the ligand binding subunit of the frog brain KAR is contained in the Mr = 48,000 proteins. Immunoblots from different frog tissues demonstrated that the antibody reactivity was highly concentrated in the frog nervous system with no detectable immunoreactivity observed in non-neuronal tissues. The purified KAR was radioiodinated and subjected to two-dimensional gel electrophoresis and autoradiography. A series of proteins was detected at Mr = 48,000 with isoelectric points from 5.5 to 6.3. The anti-KAR mAb and the antiserum reacted with the same group of proteins from frog whole brain after separation by two-dimensional gel electrophoresis. Peptide maps of the 125I-labeled KAR separated by two-dimensional gel electrophoresis demonstrated that the group of proteins clustered at Mr = 48,000 is homologous. mAb KAR-B1 reacted on immunoblots with a protein in rat brain with a Mr = 99,000. This protein comigrated with an unreduced form of the KAR in frog brain. It was present in rat cerebral cortex, hippocampus, and cerebellum but was not detected in thalamus, globus pallidus, or brain stem, nor was it detected in rat non-neuronal tissues. The presence of the Mr = 99,000 immunoreactive polypeptide in discrete areas of rat brain suggests that this protein may be part of a mammalian KAR or a related receptor.