The binding of zolpidem and some other benzodiazepine site ligands between the alpha 1 (a1) and gamma2 (g2) GABAA receptor subunits depends on a phenylalanine (F) residue at position 77 in the g2 subunit. Substitution with an isoleucine (I) largely abolishes zolpidem binding (Buhr et al. 1997 J Biol Chem 272 11799). The zolpidem potentiation of GABAA receptor-mediated mIPSCs is abolished in Purkinje cells expressing a1b2g2 receptors in a mouse with a F to I engineered point mutation in the g2 subunit gene (g2F77I; Cope et al. 2004 Neuropharmacology). We have examined how the effect of zolpidem is modified in g2F77I hippocampal CA1 pyramidal cells and interneurons which express additional alpha and gamma subunits. Hippocampal neurons were recorded from homozygous point mutant (g2I77/I77), wildtype (g2F77/F77) littermate and control C57BL/6 mice in whole-cell patch clamp mode. Zolpidem (1uM), in the presence of kynurenic acid (3mM) and TTX (0.5-1uM), significantly (P<0.05) potentiated both the peak amplitude and weighted decay of mIPSCs in pyramidal cells of C57BL/6 (119% and 161%, respectively) and g2F77/F77 (118% and 157%) mice. Similar effects were detected in biocytin-labelled interneurons of C57BL/6 (135% and 158%) and g2F77/F77 (115% and 140%) mice. In contrast to g2I77/I77 Purkinje cells, zolpidem slightly but significantly potentiated the weighted decay of mIPSCs in g2I77/I77 pyramidal cells (109%) as well as in interneurons (109%). In g2I77/I77 mice zolpidem decreased mIPSC peak amplitude in pyramidal cells (95%). Zolpidem at 100nM also significantly increased the weighted decay of mIPSCs in g2I77/I77 pyramidal cells (112%), although this was much smaller than in C57BL/6 and g2F77/F77 mice. We suggest that these residual effects of zolpidem are due to binding to a1bxg1 receptors, or that the F77 residue is less important for zolpidem binding at a2/3bxg2 receptors. The g2F77I mice may help clarify the roles of g1 subunit containing receptors.
Residual effects of zolpidem on GABAA receptor-mediated mIPSCs of hippocampal neurons in F77I gamma2 subunit point mutant mice.
OBERTO, Alessandra;
2004-01-01
Abstract
The binding of zolpidem and some other benzodiazepine site ligands between the alpha 1 (a1) and gamma2 (g2) GABAA receptor subunits depends on a phenylalanine (F) residue at position 77 in the g2 subunit. Substitution with an isoleucine (I) largely abolishes zolpidem binding (Buhr et al. 1997 J Biol Chem 272 11799). The zolpidem potentiation of GABAA receptor-mediated mIPSCs is abolished in Purkinje cells expressing a1b2g2 receptors in a mouse with a F to I engineered point mutation in the g2 subunit gene (g2F77I; Cope et al. 2004 Neuropharmacology). We have examined how the effect of zolpidem is modified in g2F77I hippocampal CA1 pyramidal cells and interneurons which express additional alpha and gamma subunits. Hippocampal neurons were recorded from homozygous point mutant (g2I77/I77), wildtype (g2F77/F77) littermate and control C57BL/6 mice in whole-cell patch clamp mode. Zolpidem (1uM), in the presence of kynurenic acid (3mM) and TTX (0.5-1uM), significantly (P<0.05) potentiated both the peak amplitude and weighted decay of mIPSCs in pyramidal cells of C57BL/6 (119% and 161%, respectively) and g2F77/F77 (118% and 157%) mice. Similar effects were detected in biocytin-labelled interneurons of C57BL/6 (135% and 158%) and g2F77/F77 (115% and 140%) mice. In contrast to g2I77/I77 Purkinje cells, zolpidem slightly but significantly potentiated the weighted decay of mIPSCs in g2I77/I77 pyramidal cells (109%) as well as in interneurons (109%). In g2I77/I77 mice zolpidem decreased mIPSC peak amplitude in pyramidal cells (95%). Zolpidem at 100nM also significantly increased the weighted decay of mIPSCs in g2I77/I77 pyramidal cells (112%), although this was much smaller than in C57BL/6 and g2F77/F77 mice. We suggest that these residual effects of zolpidem are due to binding to a1bxg1 receptors, or that the F77 residue is less important for zolpidem binding at a2/3bxg2 receptors. The g2F77I mice may help clarify the roles of g1 subunit containing receptors.
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