The dual role of N-methyl-D-aspartate receptors (NMDARs) in the normal and abnormal functioning of the retina imposes important constraints on possible therapeutic strategies aimed at ameliorating NMDAR-mediated insults to retinal ganglion cells such as ischemia and glaucoma. Block of NMDAR overactivity must be achieved without interference with normal function. Two groups of drugs show promise for safe but effective pharmacological intervention to curtail excessive activity of the NMDAR: (i) memantine, a use-dependent and uncompetitive antagonist that is an NMDA open-channel blocker, and (ii) nitric oxide (NO)-related species (e.g. provided by nitroglycerin) that interact with redox modulatory site(s) on the NMDAR. Preliminary results demonstrate that 1) glutamine/arginine/asparagine (Q/R/N) sites in the second membrane spanning (M2) domain of NR1 and NR2 subunits of the NMDARs strongly affect memantine binding 2) there are three kinetically distinct components of redox modulation on NR1a/NR2a heterometric channels that are affected by redox agents; 3) each kinetic component of redox modulation can be attributed to interaction between with a pair of Cys residues on the NR1 or NR2A subunits, and these subunits also affect modulation by Zn; and 4) one Cys residue on NR2A is mainly responsible for inhibition of NMDAR activity by NO-related species via S-nitrosylation (transfer of NO+ to thiol). To avoid systemic side effects NO-related species will be chemically linked to memantine to target the NO group to its NMDAR Cys redox site. Based on Cys residues in the redox site, a novel NMDAR subunit, NMDAR3 has been cloned from the rat retina and recently a second DNA has been cloned which encodes another NMDAR subunit, NR3b. The following aims are proposed: [1] To study the structural determinants of memantine binding in the channel pore of the NMDAR. [2] To characterize the Cys residues that underlie redox Zn and No modulation of the NMDAR and to develop combinatorial NO-memantine drugs designed to target NO group transfer to Cys residues of the NMDAR. Importantly, the novel NMDAR antagonist drugs designed here can then be developed for neuroprotection of RGCs from ischemic and glaucomatous insults. [3] To elucidate the molecular mechanism of action of NR3A whereby it decreases NMDAR-activated current. [4] To clone and characterize a second NMDAR subunit, NR3B, isolated from the rat retina.
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