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RESEARCH PROJECTS AND FINDINGS-3


3. Regulation of NMDA Receptor by Polyamines and Development of a Channel Blocker(s) of NMDA Receptor


Fig. 3-1
3-1s
Fig. 3-2
3-2s
(Click the figure, then you can see the larger one.)

Spermine has multiple effects on N-methyl-D-aspartate (NMDA) receptors, including "glycine-independent" stimulation, which is seen in the presence of saturating concentrations of glycine, and voltage-dependent block. To identify amino acid residues that are important for spermine modulation, we used site-directed mutagenesis to alter amino acids of the NR1 subunit of the NMDA receptor. Mutated subunits, expressed in heteromeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. We found that Asp170, Glu181, Glu185, Glu186, Glu297, Asp303, Glu339 and Glu342 were involved in "glycine-independent" stimulation. NR1 (D130N) mutant reduced sensitivity to ifenprodil by more than 500-fold, but had little effect on sensitivity to spermine and pH. Mutations at six other residues in this region of the NR1 subunit reduced the potency of ifenprodil. To model this region of NR1, we compared the predicted secondary structure of NR1 (residues 19-400) with the known structures of 1,400 proteins. This region of NR1 is most similar to bacterial leucine/isoleucine/valine binding protein, a globular amino acid binding protein containing two lobes, similar to the downstream S1-S2 region of glutamate receptors. We propose that the tertiary structure of NR1 (22-375) is similar to leucine/isoleucine/valine binding protein, containing two "regulatory" domains, which we term R1 and R2. This region, which contains the binding sites for spermine and ifenprodil, may influence the downstream S1 and S2 domains that constitute the glycine binding pocket (Fig. 3-1).
A number of structurally diverse compounds (memantine, MK-801 and tribenzylspermidine) act as open-channel blockers of NMDA receptors. They share discrete or overlapping binding sites within the channel. We recently found that anthraquinone (AQ)- and anthracene (Ant)-polyamines are potent antagonists of NMDA receptors. The effects of both AQ- and Ant-polyamines were reversible and voltage-dependent. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by AQ- and Ant-polyamines. These residues were located in the outer vestibule at the selectivity filter/narrowest constriction of the channel and in the inner vestibule below the level of the selectivity filter. Thus, AQ- and Ant-polyamines are new type of channel blockers of NMDA receptor; the binding site of these amines is different from that of memantine, MK-801 and tribenzylspermidine. The results with mutant NR1 and NR2 subunits were consistent with the idea that NR1(Asn616) and NR2B(Asn616), but not NR2B (Asn615) make the narrowest constriction of NMDA channel (Fig. 3-2). Experiments are in progress to develop the clinically useful channel blockers.

Last Update: 05/16/2007

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