We have previously analyzed the alpha bungarotoxin region of the acetylcholine receptor (AcChR) using six monoclonal antibodies (mAbs) directed against this portion of the AcChR. These mAbs were able to distinguish five distinct epitopes within this region. We now propose to carry out a similar analysis of new mAbs (to be produced in the Scientific Core) directed against synthetic peptides corresponding to defined sites within the primary sequence of the AcChR subunits and that crossreact with native AcChR. mAbs directed against fluorescent dyes that are capable of being attached to AcChR will also be studied in terms of their interaction with dye-labeled AcChR. Both sets of mAbs will be directed against defined epitopes within the primary structure of AcChR and will therefore represent site-specific probes. The avidity and stoichiometry of binding of those mAbs will be determined by equilibrium binding studies. The effect of mAb binding on the binding of alpha bungarotoxin, of cholinergic agonists and antagonists will be determined, as will the effect of the toxin and the ligands on mAb binding. The ability of each mAb to interfere with the binding of the others will also be assessed. The functional analysis of these mAbs carried out in this Project will complement analyses of the effects of these site- specific mAb probes on ion channel function (Projects 2 and 3) or on neuromuscular transmission (Project 4). For each mAb, these functional data will then be correlated with information on the location of its epitope in terms of both primary (Project 5) and three-dimensional structure (Project 6) of the AcChR.
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