In many neurological disorders, injury to neurons may be caused, at least in part, by overstimulation of receptors for excitatory amino acids, including glutamate, aspartate and related congeners. These neurological conditions range from acute insults such as stroke, hypoglycemia, trauma, and epilepsy, to chronic neurodegenerative states such as Huntington's disease, AIDS dementia complex, amyotrophic lateral sclerosis, and perhaps Alzheimer's disease. Glutamate is the major excitatory neurotransinitter in the brain and as such its interactions with specific membrane receptors are responsible for many normal neurological functions including cognition, memory, movement, and sensation. In addition, excitatory neurotransmitters are important in shaping the developmental plasticity of synaptic connections in the nervous system. However, in a variety of pathological conditions, including stroke and various neurodegenerative disorders, excessive activation of glutamate receptors may mediate neuronal injury or death. Olney coined the term 'excitotoxicity' for this condition, which may constitute a final common pathway for neuronal injury from diseases of diverse pathophysiology. This form of injury appears to be predominantly mediated by excessive influx of Ca2+ into neurons through ionic channels triggered by activation of glutamate receptors. An important point concerning these glutarnate receptor subtypes is that the N-methel-D-aspartate (NMDA) subtype of glutamate receptoractivated channel permits the influx of Ca2+ as well Na as+9 and overstimulation of this type of receptor is thought to be the predominant mechanism for calcium overload in neurons. Ca2+ influx triggered via NMDA receptor stimulation activates a variety of enzymes including nitric oxide synthase and the consequent production of nitric oxide. When NMDA receptors are excessively stimulated, nitric oxide may be produced in increased quantities. Under these conditions, NO- and 02'- may react to form a toxic substance called peroxynitrite (ONOO-), resulting in neuronal death In contrast, nitric oxide can be converted to a different chemical state that has just the opposite effect, protecting neurons from injury due to NMDA receptor overstimulation. The chemical state is dependent upon the removal or addition of an electron to nitric oxide, a condition that can be influenced by the presence or absence of electron donors, such as ascorbate or the amino acid cysteine. For example, with one less electron, NOmay yield a substance with NO+ (nitrosonium) character. In this form, the NO group appears to be transferred to a regulatory site on the NMDA receptor, termed the redox modulatory site. This site is comprised of sulfhydryl (-SH) groups; the reaction of -S- with NO+ to form -SNO (a process called Snitrosylation) results in decreased activity of the NMDA receptor, thus affording protection from excessive stimulation. Therefore, depending on its chemical state, the NO moiety can lead to neurodestruction or neuroprotection. These findings have lead to therapeutic approaches to decrease NMDA receptor overactivity using drugs with NO+ character, such as nitroglycerin. NMDA Redox Modulatory Site The motivating force behind the work proposed here is to characterize the cysteine residues comprising the redox modulatory site(s) of the NMDA receptor with respect to their interaction with the NO group. Work from our laboratory with recombinant NMDA receptor subunits has shown that two cysteine residues on the NMDARI subunit of the NMDA receptor are involved in redox modulation (cysteine residues 744 and 798; Fig .1, below). In collaboration with the Chait laboratory, we propose to use electrospray ionization mass spectrometry to visualize S-nitrosylatoin of the NMDARI subunit, to determine if intra- or intermolecular disulfide bond formation subsequently occurs, and to correlate these findings with the know regulatory activity of these reactions on the NMDA receptor from our patch-clamp electrophysiological studies.
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