Transmembrane proteins like the acetylcholine receptor contain portions of their structure in a lipid environment and others in media of different ionic strengths and composition inside and outside the cell. Elucidation of the roles of the outside (exocytoplasmic) sections of the protein molecule is of interest to this Project. The proposed work has two basic thrusts: 1) to assess the effects of monoclonal antibodies on protein function, 2) to identify structural sites recognized by specific monoclonal antibodies, that are effectors of channels activity. We will use the acetylcholine receptor as a prototype cationic channel for organic and inorganic cations. In these studies we synthesize and obtain a self-consistent image of molecular events for a protein- mediated membrane event integrating results from the following techniques: 1) kinetic procedures equilibrium binding, ion flux rates, fluorescence quenching stopped-flow procedures, 2) biological tools: monoclonal antibodies, 3) chemical procedures: peptide fractionation and isolation, chemical characterization of peptides, controlled proteolysis, protein chemistry.
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