George Paul Hess, A.B., Ph.D. - US grants

The mechanism of the acetylcholine receptor-controlled translocation of inorganic ions through membranes is being investigated. The problem is of interest because proteins like this receptor control the transmission of signals between nerve cells and nerve muscle cells. During the last 4 years we have developed methods and techniques (with a time resolution of about 5 msec) which allow kinetic measurements of ion translocation to be made in receptor-containing membrane vesicles obtained from the electric eel. The intrinsic rate constant, J, for the receptor-controlled ion translocation has been determined and ion translocation rates have been related to (i) protein isomerization rates, (ii) the equilibrium constants of the ligand binding process, and (iii) the channel opening equilibrium. The minimum model based on our measurements can be treated quantitatively and accounts for the ion translocation process over the whole range of acetylcholine (5,000x) and carbamylcholine (200x) concentration range investigated. We now plan to (i) complete our studies of receptor function in membrane vesicles in the msec time region by investigations which appear to be of physiological interest and which have been only partially resolved in studies with cells: (a) the receptor-controlled flux of Ca2+ and the effect of Ca2+ on receptor-controlled translocation of Na+ and K+, (b) the effects of transmembrane voltage, inorganic ion concentration, and temperature on the ion translocation process; (ii) develop techniques which allow chemical kinetic measurements with vesicles to be made in the M usec time region. Involved are reagents which can be photolyzed (in the M usec time region) to acetylcholine or its analogs, and involve Beta-carotene as an indicator of transmembrane voltage changes in the sub M usec region and resonance Raman spectroscopy to detect changes in membrane potential; (iii) to determine the rate constants of opening and closing of the transmembrane ion channels; (iv) extend investigations to other receptors both because of the importance of the molecules and to make the techniques which we have developed generally useful in investigations of the many known membrane-bound proteins which mediate the transfer of ions and molecules across membranes.

The long-term objective is elucidation of the chemical mechanism of neurotransmitter receptor-mediated reactions in the membrane of nerve and muscle cells. Techniques developed by this group enable one to study elementary steps of the reactions, information needed to understand the regulation of signal transmission between the approximately 10(12) cells in the human nervous system. Specific aims are to: 1. Determine reaction mechanisms of excitatory acetylcholine and glutamate and inhibitory GABA and glycine receptors. The knowledge gained is required to account for the integration of excitatory and inhibitory signals by a single cell. 2. Understand the modification of receptor function by therapeutic agents, abused drugs, and combinatorially synthesized RNA polymers. Receptors in cells and genetically engineered forms expressed in X. laevis oocytes will be used to understand how these compounds affect receptor mechanisms. 3. Understand the integrated action of a known circuit of neurons controlling a measurable behavioral response, pharyngeal pumping, in the nematode C elegans. Neurotransmitters secreted by specific cells and responding receptors in target cells will be identified. Health Relatedness of the Project: Neurotransmitter receptors regulate intercellular communication in the central nervous system and provide the mechanism by which environmental information is received, stored, and transmitted. They are implicated in nervous system diseases (Alzheimer's, Parkinson's) and are the targets of therapeutic agents (for instance, Prozac(TM), tranquilizers) and abused drugs. Understanding how receptor-mediated reaction mechanisms are modified is required for rational clinical treatment of diseases and the design of improved drugs. Research Design: Rapid reaction techniques, newly developed by this group and suitable for studying reactions on cell surfaces in the mu s-ms time region, will be used. Receptors are equilibrated with a photolabile, biologically inert neurotransmitter precursor before photolysis releases the neurotransmitter in mu s, thereby initiating the reaction. The resulting whole-cell current due to opening of receptor-channels is recorded and analyzed. This technique is used in combination with a cell- flow technique with a 5-ms time resolution. This approach overcomes the limitations of previous techniques with inadequate time resolution that has hampered research in this area during the last 40 years and can be used with intact single cells, oocytes, and circuits of cells.

DESCRIPTION (provided by applicant): 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.

The goal of this application is to build a cell-flow/laser flash photolysis apparatus for the measurement of receptor function directly on the surface of a single cell with a microsecond-to- millisecond time resolution. a technique with such a time resolution has not been available. The apparatus will consist of several attached parts: (1) Cell-flow device with a 20-millisecond time resolution for applying photolabile neurotransmitters to a single cell. (2) Whole-cell current recording apparatus, with associated computing, for recording and analyzing the whole-cell currents that are induced by the applied neurotransmitter. (3) Laser and associated optics for (a) determining the rate of photolysis and quantum yield of the photolabile compound, and (b) removing the photolabile protecting group and, thereby, releasing the neurotransmitter, which binds to its specific neurotransmitter and induces the flow of currents across the cell surface. Neurotransmitter release has a time resolution of 200 microseconds. The measurements can be made with any cell that carries receptors for acetylcholine, GABA, glycine, glutamate, aspartic acid, or N- methyl-D-aspartate. We have made photolabile precursors of all these compounds.

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.