Research this past year was focussed on the molecular characterization of excitatory amino acid receptors. Using probes based on protein sequence of a kainic acid binding protein purified in this laboratory, several clones were isolated from a frog brain cDNA library. The deduced amino acid sequence from the overlapping isolated clones showed that the CDNA encodes a 48 kDa protein of 470 amino acids. Scatchard analysis of (3)H-kainic acid binding to membranes of COS-7 cells transfected with the CDNA demonstrated the presence of high affinity binding, but experiments with transfected cells or Xenopus oocytes injected with mRNA did not show a physiologically active kainate receptor. A characteristic hydrophobic feature found in neurotransmitter gated ion channel subunits was conserved in the 48 kDa protein. Comparison of the kainate binding protein amino acid sequence with the sequence of a mammalian brain excitatory amino acid receptor, GluR-K1, showed a high degree of homology between the two proteins providing strong evidence that they are evolutionarily related. GluR-Kl has been demonstrated through physiological analysis to be a kainic acid receptor. Immunoaffinity purification of GluR-Kl using antipeptide antibodies, based on the deduced amino acid sequence of the protein, showed this receptor binds AMPA, but not kainic acid. An AMPA binding protein was solubilized and purified from bovine brain. The purified sites migrated as a single peak with gel filtration with an Mr = 425,000 and displayed binding characteristics like those of the membrane associated sites. With SDS electrophoresis a single major band with Mr = 114,000 was present. In situ hybridization analysis of neurotransmitter receptors in auditory nuclei of the brainstem show a variable distribution for subtypes of GABA and glycine receptors. With probes for the glycine receptor, the most intense labeling was found in the lateral superior olive and the nuclei of the lateral lemniscus, while alpha I subunit of the GABAA receptor is very abundant in the inferior colliculus and moderately abundant in the dorsal cochlear nucleus.
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