The objective of the research described here is elucidation of the molecular topology of the family of ligand-gated ion channels (i.e.,glutamate receptors) which mediate most fast synaptic transmission in the CNS. Despite widespread acceptance of the newest model of glutamate receptor topology, the regions flanking the proposed hairpin structure for the pore-forming region (i.e., M2) have not yet successfully been demonstrated to reside on the same side of the cell membrane in a functional protein. Using the NR1 subunit of the N-methyl- D-aspartate (NMDA) receptor as a prototype, the hypothesized existence of the M2 hairpin structure will be tested. In the flanking regions, specific markers (e.g., phosphorylation or protease-sensitive sites) will be introduced via mutagenesis and tested during single channel patch clamp records in order to localize their orientation in functional receptors. The benefits of performing such a study on the essential NMDA receptor subunit are numerous. The receptor is believed to mediate neuronal degeneration in a variety of pathologic conditions ranging from stroke to epilepsy. Additionally, it has been proposed to play a fundamental role in long-term memory formation. Finally, a rational drug design for pharmacologic manipulation of this potential target would require knowledge of the amino acid residues accessible from the extracellular space.
