| Co-Investigator(Kenkyū-buntansha) |
SHIRAHARA Akira Josai University, Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (50150107)
KASHIWAGI Keiko Chiba University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (80169424)
KAKINUMA Yoshimi Chiba University, Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (80134394)
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| Budget Amount *help |
¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 2000: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1999: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
1.There are complex interactions between spermine, protons, and ifenprodil at N-methyl-D-aspartate(NMDA)receptors. Spermine stimulation may involve relief of proton inhibition, whereas ifenprodil inhibition may involve an increase in proton inhibition. We studied mutations at acidic residues in the NR1 subunit using voltage-clamp recording of NR1/NR2B receptors expressed in Xenopus oocytes. Mutations at residues near the site of the exon-5 insert, including E181 and E185, reduced spermine stimulation and proton inhibition. Mutation NR1(D130N)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 and, in some cases, the maximum effect of ifenprodil. These mutants did not affect sensitivity to pH, glutamate, glycine, or other hallmark properties of NMDA channels such as Mg^<2+> blodk and Ba^<2+> permeability. Residues in this region presumably fo
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rm part of the ifenprodil-binding site. 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.
2.The effects of aminoglycoside antibiotics on NMDA receptors were studied using voltage-clamp recording of recombinant NMDA receptors expressed in Xenopus oocytes. A number of aminoglycosides were found to potentiate macroscopic currents at heteromeric NR1_A/NR2B receptors, but not at NR1_A/NR2A, NR1_A/NR2C, NR1_A/NR2D or NR1_B/NR2B receptors. The degree of potentiation had a rank order neomycin B > paromomycin > gentamicin C > geneticin > kanamycin A > streptomycin. Potentiation was not seen with kasugamycin and spectinomycin. The degree of stimulation paralleled the number of the amino groups in the aminoglycosides. The stimulatory effects of aminoglycosides were more pronounced at subsaturating concentrations of glycine and at acidic pH, similar to the stimulatory effects of spermine. We measured the effects of aminoglycosides at mutant NMDA receptors to determine which amino acid residues in NMDA receptor subunits are involved in stimulation. Mutations that reduced or abolished spermine stimulation also reduced stimulation by aminoglycosides. Several aminoglycosides produced a weak voltage-dependent block of NMDA receptors, but the degree of inhibition did not appear to correlated with the number of amino groups in the molecule. The results suggest that aminoglycosides having more than three amino groups have stimulatory effects that are mediated through the spermine-binding site on NMDA receptors.
3. Polyamine derivatives of anthraquinon were found to be NMDA receptor antagonists. Less
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