9631969 Hicks Advanced techniques in neuroscience and spectroscopy will be utilized to study the n-methyl d-aspartate (NMDA) neuronal receptor. This receptor is involved in the long-term potentiation, memory and developmental structuring; its central role in long-term changes in the brain make its study of particular value. It binds several agonists including NMDA, glutamate and the drug PCP. The receptor is known to be a ligand-gated ion channel, and to consist of two protein subunits, both of which have been cloned. The NR1 subunit has a predicted mass of 103 kDa, and is thought to consist of a large extracellular N-terminus, three trans-membrane-spanning domains, a hairpin domain within the membrane and an intracellular carboxy terminus. The exact structure of the protein and the agonist binding sites are not yet known. The objectives of this proposal are to use site-directed mutagenesis to identify residues that control glutamate and PCP binding. Mutationally altered receptors will be expressed in Xenopus oocytes and analyzed functionally by the two electrode voltage clamp technique. In addition, surface plasmon resonance spectroscopy will be used to study the binding of glutamate, PCP and PCP analogs to the mutant receptors, providing additional kinetic information about adsorption and desorption processes. The long term goal is to design experiments utilizing spectroscopic laser methods that will address questions regarding the structures of native and mutant receptors and bound ligand/receptor complexes. Previously, the PI has developed sensitive methods for measuring circular-dichroism spectra at interfaces (SHG-CD). Circular dichroism spectroscopy is a well known method for the analysis of protein secondary structures; the laser method SHG-CD has the added advantage of surface sensitivity. The PI proposes to spend one year working with Dr. Suzanne Zukin, Professor, and Dr. Michael Bennett, Professor and Chair, in the Department of Neuroscience at the Albert Einstein Colle ge of Medicine. The PI is a physical chemist who has 12 years of experience developing laser methods that probe molecular interfaces in situ. She has studied small molecule surface chemistry and the adsorption of polypeptides at crystalline surfaces. Dr. Zukin has been actively engaged in research in the area of neuroreceptors for twenty years, focusing on the molecular biology and physiology of the excitatory amino acid and opiate receptor systems of the central nervous system. Dr. Bennett's work in neuroscience research spans 40 years, with current interests in ligand-gated channels mediating excitatory postsynaptic potentials, studies using electrophysiological methods. The sabbatical research will allow the PI to learn manipulation of cloned genes in, handling and microelectrophysiological measurement of Xenopus oocytes, cells which are large and possibly amendable for direct laser study. Specific techniques here include engineering of mutant receptor constructs, expression and functional analysis of receptors by electrical recording of cells and by surface plasmon resonance spectroscopy. %%% The passing of information from one brain cell to the next allows for all of brain activity, such as a memory, thought and responses to our senses like vision, hearing, feeling, etc. This transmission of information is accomplished by the transport of molecules called neurotransmitters from one cell surface to the next. A cell's surface is composed of fatty acids (lipids) and many other components including proteins called receptors. Receptors receive the incoming neurotransmitter molecules in what is usually envisioned as a "lock and key" mechanism. That is, the shape of the neurotransmitter allows it (the key) to "fit" into the receptor (the lock). The details of the geometry and the forces around the lock and the key hold the secret of the transmission of signals in the brain. Drug design for brain disorders relies heavily on this knowledge. Dr. Hicks will apply advanced laser methods to study one particular neural receptor called the NMDA receptor, which is responsible for memory and long-term changes in the brain. The laser based methods (second harmonic generation (SHG) and surface plasmon resonance (SPR) spectroscopy) will allow details of the transmitter/receptor interaction to be measured. These laser methods have proven to be surface sensitive, but are new in the field of biophysics. For example, SHG has not yet been applied to a working cell membrane. This award supports a novel interdisciplinary approach to an important issue in brain chemistry. ***
