This research will investigate the interaction of the agonist nicotine and the neurotropic rabies virus with the nicotinic acetylcholine receptor (AChR) subunit which contains a major cholinergic-binding site. Emphasis is placed on investigation of neuronal nicotinic AChRs which are not as well understood as the muscle type AChR. The agonist-binding site on the neuronal alpha- 4 subunit will be identified and the binding of [3H]nicotine and [3H]cytisine to synthetic peptides and fusion proteins of this region characterized. To study structure-function relationships, residues within the major agonist-binding site will then he substituted by oligonucleotide-directed mutagenesis with amino acids from alpha-1, alpha- 2, alpha-3, alpha-5, and alpha-7 subunits and effects on binding of nicotine, cytisine, alpha-bungarotoxin, and kappa-bungarotoxin measured. In this manner, the residues responsible for binding and the differential sensitivities of neuronal alpha-subunits to agonists and antagonists will be identified. Additional mutations will he performed to investigate the functional contributions of highly conserved Tyr-190 and Tyr-198. Amino acids labeled by nicotine and cytisine will he identified by sequence analysis. The effect of the mutations on conformation of the peptides will he determined by circular dichroism spectroscopy. Characterization of structure-function relationships of nicotine interaction with different receptor subtypes can identify potential targets for therapeutic intervention in addiction and in diseases such as Alzheimer's disease in which cholinergic function is disrupted. The interaction of rabies virus, a rhabdovirus whose glycoprotein bears a region with an amino acid sequence similarity to neurotoxins, with muscle and brain AChRs will he investigated. It will he determined whether the AChR is essential for rabies virus infection by testing whether transfection of a nonpermissive cell line with AChR subunit cDNAs confers susceptibility to infection. she binding site on the receptor will he localized by preparing deletion mutants of the AChR alpha1 subunit and testing their ability to serve as functional virus receptors. Studies will be undertaken to determine whether rabies virus utilizes central nicotinic AChRs as host cell receptors. Radioautographic localization of rabies virus binding sites in the brain will he compared with the patterns of expression of different nicotinic AChR alpha subunit subtypes. The effect of synthetic peptides and fusion proteins of the AChR on rabies virus infection of muscle and central neurons in primary culture will be tested. Identification of host cell receptors for viruses and characterization of the virus-binding site on the receptor allow design of antiviral agents that mimic the receptor and inhibit the attachment stage of the viral infectious cycle.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Neurological Sciences Subcommittee 1 (NLS)
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Yale University
Anatomy/Cell Biology
Schools of Medicine
New Haven
United States
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