STRUCTURE/FUNCTION ANALYSIS OF ACCHR EPITOPES

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The central theme of this Program Project is to use monoclonal antibodies (mAbs) directed against defined oligopeptides within the subunits of the nicotinic acetylcholine receptor (AcChR) as site-specific probes of AcChR structure (both primary and tertiary structure) and AcChR function. Special attention will be paid to mAbs whose binding interferes with the function of this transmembrane glycoprotein neurotransmitter receptor. Projects 1-4 will assess the effects of the mAbs on particular aspects of AcChR function: Project 1 - ligand binding, Project 2 - ion fluxes by stopped flow technique; Project 3 - ion channel function by patch clamp technique; Project 4 - neuromuscular transmission in vitro and in vivo. Projects 5 and 6 will localize the epitopes of these mAbs within the AcChR structure: Project 5 - production of rationally chosen synthetic oligopeptides mimicking specific sites within AcChR subunits, assessment of the contribution of glycosyl moieties to mAb epitopes, Project 6 -identification of bound mAbs on the low resolution three-dimensional model of AcChR by both small angle X-ray diffraction and high resolution electron microscopy, as well as localization of fluorescent probes. The Scientific Core will produce new mAbs directed against the peptides produced in Project 5 and will provide purified, characterized mAbs for the various projects. In Project 4, the peptides will be used to possibly develop a new treatment modality for experimental myasthenia gravis. This Program Project represents a collaborative effort of immunologists, biochemists, electrophysiologists, peptide chemists, and physical/structural chemists in which the information from the various projects, taken together, will allow for mapping of various sites in the primary sequence of AcChR, especially functionally important sites, onto the three-dimensional model.