The binding of neurotransmitters and inhibitors to membrane-bound receptor proteins, the resulting receptor-controlled flux rates and the rates of receptor inactivation (desensitization) are important in determining the transmission of signals between cells, and constitute some of the limits within which the nervous system can function. Investigations of the relationship between ligand-binding processes, receptor-controlled flux rates, and rates of receptor desensitization, under well-defined conditions and with a time resolution of 5 ms have become possible with membrane vesicles containing acetylcholine receptor from the electric organ of E. electricus using methods and techniques which we developed. It is now planned to extend these investigations to receptors of the central nervous system (CNS). The techniques to make these investigations are not available and will be developed: (i) New approaches generally suitable for kinetic measurements of receptor function on cell surfaces, involving the synthesis of photolabile protecting groups for amino groups, because these exist on many different neurotransmitters. The photolysis of these compounds in situ in the Mus time region turns on the receptors, allowing synchronous measurements of the time course of the onset of integrated cell currents. This technique is being developed to provide (i) mechanistic information about CNS receptors not attainable previously, and (ii) access to a previously inaccessible time region, which allows kinetic investigations of elementary steps in the formation of transmembrane channels. (ii) An optical detection system, which may become generally useful in identifying the type of receptors associated with defined cells in cell cultures. The cells are equilibrated with a fluorescent label that is quenched by Cs+. Cs+ is translocated rapidly across the cell membrane when receptor channels are activated by specific chemical signals. Quantitative fluorescence microscopy will be used to locate cells with altered fluorescence. This technique will be used to locate acetylcholine and other receptors in dissociated CNS cells. (iii) A new expression system that may become generally useful for investigation of the structure and function of neuroreceptors, which exist only in low concentrations in CNS cells, will be investigated. It will first be determined if the subunits of the Torpedo acetylcholine receptor can be produced, isolated and assembled in a functional form in artificial membranes or synthesized by the yeast cell. Rational clinical treatment of diseases caused by receptor dysfunction is expected to be aided by all the proposed investigations.
