The first goal of this research project is to extend a newly developed laser-pulse photolysis technique, which at present allows one to investigate the function of he acetylcholine receptor in a muscle cell in the us time region, to excitatory (glutamate, aspartate, N-methyl-D aspartate (NMDA) and inhibitory (glycine and gamma-aminobutyric acid) receptors in mammalian central nervous system (CNS) cells. The techniques requires pre-equilibration of receptor-containing cells with a photolabile inert precursor of a neurotransmitter (caged neurotransmitter), release of the neurotransmitter by a laser pulse, and recording and analyzing the whole-cell current due to the opening of receptor channels. The caged neurotransmitter must be photolyzed in us and be biologically inert. The following will be done: (1) Synthesize caged neurotransmitter in which the carboxyl group is blocked by a new photolabile protecting group; caged glycine is photolyzed within 2 us or about 50 times faster than caged carbamoylcholine (an acetylcholine analog) and about 500 times faster, and with a quantum yield-4x larger than cage neurotransmitter presently available. (2) Characterize the new compounds by NMR, CNH analysis and/or mass spectroscopy. (3) Determine photolysis rates and quantum yield using a spectrophotometer with 1-us time resolution. (4) Determine if the cage compounds and their photoplays products have deleterious effects on receptor function and/or the cells, using a cell-flow method with a 10-ms time resolution. The second goal is to determine: (1) The channel-opening rates of CNS receptors (particularly the NMDA receptor), essential for understanding the mechanism of the reaction by which a receptor can initiate transient transmembrane voltage changes and, thereby, signal transmission between cells. (2) The mechanisms by which therapeutic agents and abused drugs affect receptor function (e.g. MK-801, a death and cocaine poisoning). The new technique for making chemical kinetic measurements on a single cell with a us time resolution give previously unattainable information about how neurotransmitter receptors in the CNS function. Neurotransmitter receptors on cell surfaces regulates intercellular communication in the CNS, and provide the mechanism by which environmental information is received, transmitted, transduced, encoded. and stored. The receptor proteins play a role in diseases caused by receptor malfunction and also medicate the effects of many clinically important compounds, for example tranquilizers, and abused drugs such as cocaine.
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