The objective is to understand the mechanism by which neurotransmitter receptors regulate signal transmission between the billions of cells in the nervous system, and the effects of neurological diseases, therapeutic compounds, and abused drugs on the mechanism. Newly developed rapid, pre-steady state kinetic techniques for investigating cell surface receptors in the microsecond-to-millisecond time domain will be used. Photolabile inert precursors of the neurotransmitter (caged neurotransmitters) are equilibrated with cell surface receptors. Photolysis generates the neurotransmitter within microseconds, initiating the binding of neurotransmitter to receptors in a time rapid compared to receptor-channel opening. The resulting whole-cell current, a measure of the concentration of open channels, can then be measured in the same time region, to determine receptor: ligand binding constants, channel-opening and -closing rate constants, and the effects on these constants of drugs and nervous system diseases, and to confirm our interpretation of the mechanism by predicting the action of compounds not previously tested. At present ultraviolet light is used in the photolysis reaction. We will develop precursors that are photolysed in the visible wavelength region with adequate quantum yield in the microsecond time region and that are biologically inert before photolysis, making the method more efficient and easier to use. Use of visible light avoids photodamage to cells/receptors, substantially increases the number of measurements made with each cell, thereby reducing experimental error and the time needed to make the measurements, and permits the use of simpler and less expensive light sources. Additionally, for precursors with low quantum yields, much higher energy light can be used at wavelengths at which cellular components do not absorb light. We will develop new photolabile precursors of the neurotransmitters GABA and serotonin and of an anionic indicator for monovalent cations. Caged GABA will be used to determine the difference in the mechanism between normal GABA(A) receptors and a mutant form found in some forms of epilepsy (a disease affecting 40 million people worldwide) and to determine if the mechanism indicates how one can correct the defect in the mechanism of the epileptic receptor. Caged serotonin will be used in studies of the serotonin 5HT3 receptor mechanism and its inhibition. The caged indicator will be used in a screening assay of neurotransmitter receptor ligands.
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