Elucidating the chemical mechanism of neurotransmitter receptor-mediated reactions in the membrane of nerve and muscle cells is the objective of this application. The reactions control and integrate communication between cells of the nervous system. On binding a neurotransmitter, receptors form (i) transiently open transmembrane channels in mus to ms, and (ii) desensitized (temporarily inactive) forms in the ms time region. The reactions lead to a transient transmembrane voltage change, triggering signal transmission from one cell to another. the change is determined by (i) the concentration of inorganic ions on both sides of the membrane, (ii) the number of ions passing through the open channel per unit time, and (iii) the time-dependent concentration of the open channel. While (i) and (ii) are readily determined by available techniques, the specific aims of this application concern (iii). Intensive investigations of the mechanism, mainly of the archetypal nicotinic acetylcholine receptor, have been hampered by the inability to measure receptor-mediated reactions in the mus to ms time region. Cell- flow and laser-pulse photolysis of caged neurotransmitters, techniques newly developed by this laboratory, open up this time domain. They will be combined with genetic engineering and single-channel current recordings to determine: 1. The chemical mechanism of reactions mediated by the inhibitory gamma- aminobutyric acid (GABA) and glycine and excitatory glutamate receptors in cerebral cortical cells, and by the excitatory Torpedo acetylcholine receptor in transfected fibroblasts. What is the reaction pathway leading from free receptor and unbound neurotransmitter to transient open-channel and desensitized forms of four structurally related receptors? Knowledge of the pathway and associated constants enables one to predict the concentration of open receptor-channels as a function of neurotransmitter concentration and length of exposure to it. 2. The mechanism of inhibition of the acetylcholine receptor and modulation of GABA receptor activity. The general question is: How does inhibition or modulation of a receptor affect its chemical mechanism? How do local anesthetics (procaine), abused drugs (phencyclidine), and acetylcholine itself inhibit the acetylcholine receptor? Similarly, how is GABA receptor activity modulated by benzodiazepines (tranquilizers) that may also occur endogenously, or by a specific toxin? 3. An integrated, consistent mechanism for signal transmission in mammalian brain cells will be proposed, and refined by computer simulation. It will be tested experimentally by measuring voltage changes in a single cell after excitatory (cation-conducting) and inhibitory (anion-conducting) receptors are activated. Preliminary results indicate that elucidating receptor mechanisms is now an achievable goal, providing a basis for (i) the rational design and testing of therapeutic drugs, (ii) understanding the effects of abused drugs, and (ii) understanding changes in the mechanisms relevant to nervous system function.
