| Co-Investigator(Kenkyū-buntansha) |
WILLIAMS Keith University of Pennsylvania, School of Medicine Associate Professor, 医学部, 準教授
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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| Research Abstract |
1. We have previously found that several acidic residues in the NR1 subunit influence sensitivity to spermine and protons. To look for additional residues that are involved, we studied mutations at all of the extracellular acidic residues in the NR1 subunit using voltage-clamp recording of NR1/NR2B receptors expressed in X enopus oocytes. Mutations at residues near the site of the exon-5 insert, including E181 and E185, and at some downstream residues, reduced spermine stimulation and proton inhibition without affecting voltage-dependent block by spermine. In some cases the effects of these mutants were additive. For example, a double NR1 (E181Q, E185Q) mutant had a larger -effect than each individual mutant. Some of the acidic residues may contribute to binding sites for spermine or protons, and others may act to couple binding to channel gating.
2. The effects of aminoglycoside antibiotics on N-methyl-D-aspartate (NMDA) receptors were studied using voltage-clamp recording of recombina
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nt NMDA receptors expressed in X enopus oocytes. A number of aminoglycoside antibiotics were found to potentiate macroscopic currents at heteromeric NR1A/NR2B receptors, but not at NR1A/NR2A, NR1A/NR2C, NR1A/NR2D and NR1B/NR2B receptors. The dugree of potentiation with 200 mu M antibiotic 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. 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. The results suggest that aminoglycosides have stimulatory effects that are mediated through binding to the spermine binding site on NMDA receptors.
3. A number of mono-, di- and tri-benzyl polyamines, having benzyl substitutions on the terminal or central amino groups, inhibited responses of NR1/NR2 receptors in oocytes voltage-clamped at -70 mV.Among the most potent compouncds was N^1, N^4, N^8 -tri-benzyl-spermidine (PB-3-4), which had an IC_<50> value of 0.2 muM.
TB-3-4 was strongly voltage dependent. At a concentration of 10 muM, TB-3-4 had no effect on ci-amino-3-hydroxy-5-methyl-4-isoxazolepropionic aicd receptors expressed from the GluR1 subunit, indicating that TB-3-4 is a selective NMDA antagonist. Less
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