Nicotinic acetylcholine receptors of neurons are poorly characterized in comparison with the nicotinic receptors of skeletal muscle. A major reason for this is that snake venom alpha-neurotoxins, which have been valuable probes for muscle nicotinic receptors, are often completely ineffective at neuronal nicotinic receptors. Recently, a related family of snake neurotoxins has been described. The kappa-neurotoxins show considerable sequence homology (approximately-50%) with the alpha- neurotoxins, but have an opposite selectivity for nicotinic receptors. Kappa-Neurotoxins are potent antagonists of neuronal nicotinic receptors in avian and murine autonomic ganglia, but bind with much lower affinity to nicotinic receptors of muscle. Radiolabeled kappa-neurotoxins have been used to distinguish physiologically-defined nicotinic receptors from ganglionic alpha- neurotoxin sites, which at present have no known function. In this proposal, two kappa-neurotoxins (kappa-bungarotoxin and kappa-flavitoxin) will be used in electrophysiological, biochemical and morphological studies designed to characterize neuronal nicotinic receptors. For the first time, the effects of kappa- neurotoxins on central vertebrate neurons will be determined. Intracellular recordings will examine the electrophysiology of neuronal nicotinic receptors in slices of chick optic lobe and rat locus coeruleus. Binding and localization studies will be done in chick optic lobe, where three different nicotinic sites have been detected. Kappa-Bungarotoxin binds to at least one of these sites with high affinity, but it is presently not known which of these nicotinic sites is involved in physiological nicotinic responses. Autoradiographic studies using radiolabeled kappa-bungarotoxin, alpha-bungarotoxin, nicotine and acetylcholine will localize these sites in the optic lobe. Binding experiments will compare the properties of the biochemically-defined sites with those of physiologically-defined nicotinic receptors. The biochemistry of kappa-neurotoxins will be further defined to gain information about the active sites of neuronal nicotinic receptors. Synthetic peptides based on the known amino acid sequences of kappa-neurotoxins will be tested for activity at neuronal receptors, with the goal of defining the critical structural differences between kappa-neurotoxins and alpha- neurotoxins which mirror the structural differences between neuronal and muscle nicotinic receptors. Several disease states involve alterations in neuronal cholinergic function. A marked decrease in cholinergic transmission has been observed in patients suffering from Alzheimer's disease and Huntington's disease. By further characterizing cholinergic neurotransmission, the experiments proposed in this study may provide a better understanding of these diseases.
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