FUNCTIONAL PROPERTIES OF NEURONAL NICOTINIC RECEPTORS

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Nicotinic acetylcholine receptors (nAChRs) in the central nervous system are poorly understood in comparison with the nicotinic receptor found in muscle and electric tissue. In contrast to the muscle receptor, receptors in the central nervous system are genetically and pharmacologically quite heterogeneous. Various neuronal nAChR subunits have been isolated, and certain combinations of these subunits produce functional nAChR when expressed in frog oocytes. Pharmacological studies provide evidence that subtypes of functional nAChR exist in situ as well. The goal of this proposal is to characterize functional neuronal nAChR subtypes expressed in situ and to distinguish receptor subtypes on the basis of pharmacological and electrophysiological properties. The approach taken to examine in situ receptors is two-fold. First, intracellular electrophysiological recordings will be made in selected nuclei of chick and rat brain. The chosen nuclei are likely to have different subtypes of functional nicotinic receptors based on the results of autoradiographic and in situ hybridization studies of nicotinic receptors. The second approach involves culturing of selected central neurons from chick and rat, and using patch clamp electrophysiological recording to characterize nicotinic receptors expressed by these cells. The properties of these in situ nAChRs will be directly compared with those of a number of genetically defined nAChR subtypes which will be expressed in frog oocytes or cell lines. Subunits from chick, rat and human brain will be examined by patch clamp electrophysiology. An important pharmacological tool to be used in these experiments is the family of snake venom toxins known as the kappa-neurotoxins (K-Toxins). The best characterized K-Toxin, kappa-bungarotoxin, shows widely different affinities for various neuronal nAChR subtypes. It is thus a useful probe for defining functional receptors in situ. Three additional K-Toxins purified in this laboratory will also be examined as potential selective neuronal nAChR antagonists. Evidence from oocyte experiments implies that a snake venom component recognizes certain receptor subtypes with higher affinity than does kappa-bungarotoxin. The effects of several fractions of venom will therefore also be examined in these experiments. Several human disease states involve alterations in neuronal cholinergic function. A marked decrease in cholinergic transmission has been observed in patient's suffering Alzheimer's disease and Huntington's disease. In addition, the major addictive component in cigarette smoke is nicotine, which produces its stimulatory effect via central nicotinic receptors. By characterizing central nicotinic receptor subtypes, the experiments proposed in this study may provide a better understanding of these diseases.