The goals of this research project are two-fold: (I) To relate manifestations of nervous system function to the underlying biochemical mechanism of neuronal receptors by measuring the rate and equilibrium constants of the reactions involving the receptor proteins; the modification of the mechanism by biologically and clinically interesting compounds and in nervous system dysfunction will also be investigated. (II) To develop new chemical kinetic approaches necessary for the proposed studies which require measurements of receptor function on cell surfaces with a us to ms time resolution. Techniques with this time resolution have not been available. The following will be used: 1. A cell flow technique with a 20-millisecond time resolution for chemical kinetic investigations of the reactions mediated by the receptors acetylcholine, gamma-aminobutyric acid (GABA), glycine, glutamate, and aspartic acid. 2. A whole cell current recording technique and the associated computer programs for recording and analyzing the whole cell currents that arise in the cell flow technique. 3. Synthesis of photolabile inert precursors of the neurotransmitters listed in (1); after photolysis the compound liberated activities specific receptors. 4. Laser pulse photolysis in combination with analytical techniques to determine the rate of photolysis and the quantum yield of the photolabile precursors. 6. Laser pulse photolysis combined with a whole cell current recording technique and use of photolabile neurotransmitter precursors equilibrated with receptors on a cell surface in order to measure the kinetics of elementary steps in receptor-mediated reactions, with a time resolution of 200 museconds. The significance of this research (1) extends to a broad range of membrane proteins that (i) mediate communication between cells involved in the function of the nervous system (perception, learning, adaptation), (ii) play a role in diseases caused by receptor malfunction, and (iii) mediate the effects of many clinically important compounds, and (2) consists of developing new techniques for making chemical kinetic measurements on cell surfaces with a musecond to msecond time resolution that has previously not been available.
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