Toward an improved understanding of the role of nicotinic acetylcholine receptors (nAChR) in development and in congenital or degenerative disorders of the mammalian CNS, the following studies are proposed. Biochemical studies will be continued on sites in mature rat brain subcellular fractions that interact with putative nAChR-specific probes. These studies shall involve the use of curaremimetic neurotoxins, antibodies raised against nAChR from the electric organ of ray or eel, and affinity reagents that specifically label nAChR in muscle and electric tissue. These probes for nAChR will be used in established ligand binding assays, affinity labeling protocols and affinity purification procedures.
The aim of these studies is to elucidate the molecular features of and interrelationships between binding sites for putative nAChR probes in the mammalian CNS. Continued maintenance of the human medulloblastoma clonal line, TE671, in cell culture is proposed. Levels of expression by cultured TE671 cells of toxin and affinity reagent binding sites and nAChR-like antigenic determinants will be quantitated and compared by use of established radioligand binding and immunoassays. The objectives of these studies are to assess the relative density of ligand and antibody binding sites on the TE671 continuous line. Molecular features of sites that are affinity purified or affinity labeled by interaction with putative nAChR probes will be elucidated. Isotopic ion efflux studies have been established and will continue to be used to detect the functional response of the ensemble of physiologically-relevant nAChR on TE671 cells. Detailed investigation of small ligand, toxin and antibody actions on nAChR functional responses will be undertaken toward elucidation of the relationship between functional nAChR and binding sites for putative receptor probes.
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