EFFECTORS OF FUNCTIONAL ORGANIZATION OF TRANSMEMBRANE PR

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Many cationic channels in skeletal and heart muscle are transmembranous proteins. As such they contain structural elements for recognition of the channel opener messenger as well as for modulation of the open and close states for organic and monovalent and divalent cations. Transmembrane proteins 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 different sections of the protein molecule is of interest to this project. The proposed work is for 4 basic thrust areas: 1) role of lipids or protein conformation and function, 2) effect of monoclonal antibodies on protein function, 3) identification of structural sites for specific monoclonal antibodies effectors of channels function and 4) study of effects of other protein membrane on states related to channel function. The proposed work will use the acetylcholine receptor as prototype of cationic muscle 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 using the following techniques: 1) physical tools, differential scanning calorimetry, fluorescence dynamics and FT-infrared spectroscopy, 2) kinetic procedures, equilibrium binding, ion flux rates, fluorescence quenching stopped-flow procedures, 3) biological tools, membrane reconstitution, monoclonal antibodies, myoblasts cell culture and membrane fractionation, 4) chemical procedures, peptide fractionation and isolation, chemical characterization of peptides, labeling with affinity ligands, reductive methylation, controlled proteolysis, protein chemistry. These different lines of input are integrated within one single laboratory environment.