The dual role of NMDA receptors in the normal and abnormal functioning of the nervous system imposes important constraints on possible therapeutic strategies aimed at ameliorating ro abating developmental disorders and neurological disease. Based on our groups previous work, two groups of drugs are likely to show promise for safe but effective pharmacological intervention to curtail excessive activity of the NMDA receptor: (i) memantine, a use-dependent and uncompetitive antagonist that is an NMDA open-channel blocker, and (ii) nitric oxide (NO)-related species (provided by nitroglycerin) that interact with redox modulatory sites on the NMDA receptor. In our preliminary results, we have demonstrated the following: 1) the glutamine/arginine/asparagine (Q/R/N) sites in the second membrane spanning (M2) domains of NMDAR1 and NMDAR2 subunits strongly affect memantine binding; 2) there exist three kinetically-distinct components of redox modulation on NR1a/NR2A heteromeric channels that are affected by redox agents; 3) each kinetic NR2A component or redox modulation can be attributed to interaction with a pair of cysteine residues on the NR1 or NR2A subunits and these subunits also affect modulation by Zn; 4) one cysteine residue on NR2A is predominantly responsible for modulation of the NMDAR activity by NO-related species. Furthermore, we recently isolated a novel NMDAR subunit, NR3A (previously designated NMDAR-L or khi-1), from the rat CNS. Co-expression of NR1/nr2a OR b/nr3a IN Xenopus oocytes decreases unitary conductance in single-channel recordings. Conversely, NR3A-deficient mice have larger than normal NMDA-evoked currents. The new project will further study these knock-out mice. We also obtained a near full-length cDNA clone of a second novel NMDAR subunit, tentatively named NR3B since it shows high sequence identify to NR3A. We propose: [1] To study the structural determinants of memantine binding in the channel pore of the NMDAR, [2] To characterize the cysteine residues underlying redox, Zn, and No modulation of the NMDAR, [3] To elucidate the molecular mechanism of action of NR3A whereby it decreases NMDAR-activated current. [4] To clone and characterize a second novel NMDAR subunit, NR3B, in order to determine the function role that it subserves in the developing brain. Most importantly, drugs developed in Project will be used for neuroprotection from hypoxic-ischemic injury and AIDS-related neuronal damage.
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