ENDOGENOUS REGULATORS OF GLUTAMATE-ACTIVATED CHANNELS

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The role of glutamate as a transmitter at central excitatory synapses is now well established. Although these pathways are of obvious importance for the normal transfer of information throughout the brain, it has come as somewhat of a surprise that signalling at glutamate synapses is much more complex than predicted from classical studies of the neuromuscular junction. This is nowhere more apparent than in the limbic system where such diverse phenomena as learning and memory, phencyclidine (PCP)- evoked psychosis and 'excitotoxic' brain injury have all been closely linked to the activity of glutamate receptors. Thus the activity of glutamate receptors is likely to contribute to the symptoms in schizophrenia, dementing illness such as Huntington's and Alzheimer's, and to brain damage caused by prolonged seizures or stroke. In some cases, abnormalities of glutamate receptors may be causal. Transmitter released from presynaptic terminals activates two classes of postsynaptic glutamate-activated channels, selectively activated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) and NMDA (N- methyl-D-aspartate). Despite intense scientific interest in glutamate receptors, their regulation remains poorly characterized. The purpose of this project is to examine two aspects of glutamate receptor regulation in hippocampal neurons. I. Increases in intracellular calcium can lead to slow 'rundown' of the NMDA channel, and high energy phosphates counteract rundown by a mechanism that does not appear to require direct receptor phosphorylation. This downregulatory mechanism may limit calcium influx into dendritic spines and thus modulate cellular responses to synaptic stimulation. Aims 1-3 will examine the action of intracellular calcium and ATP on NMDA receptor/channels. II. Phosphorylation of postsynaptic glutamate receptors is postulated to be an important regulator of synaptic transmission. However, the rapid action of phosphatases and phosphodiesterases suggest that kinases may need to be located near a membrane substrate such as a receptor in order to be effective. In Aim 4 the hypothesis that kinase localization by specific anchoring proteins is required for phosphorylation of AMPA receptors in the postsynaptic density will be tested. Peptide inhibitors of anchoring proteins for the regulatory subunit (RII) of cAMP-dependent protein kinase will be introduced into hippocampal neurons. Whole-cell patch clamp recording and measurements of intracellular calcium in single cultured hippocampal neurons will be used. The composition of the cell cytoplasm will be controlled using intracellular perfusion and flash photolysis of "caged" compounds. Single glutamate channels will be studied in the cell-attached and inside-out configuration. Molecular methods including expression of receptor subunits in cell lines will be used to probe the regulator sites on specific receptor subunits. The results of these studies are expected to lead to more effective therapeutic strategies for altering synaptic transmission in neuropsychiatric disorders.