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. 2010 Jun 25;636(1-3):18-27.
doi: 10.1016/j.ejphar.2010.03.015. Epub 2010 Mar 19.

The point mutation gamma 2F77I changes the potency and efficacy of benzodiazepine site ligands in different GABAA receptor subtypes

Joachim Ramerstorfer  1 , Roman Furtmüller, Elisabeth Vogel, Sigismund Huck, Werner Sieghart
Affiliations

The point mutation gamma 2F77I changes the potency and efficacy of benzodiazepine site ligands in different GABAA receptor subtypes

Joachim Ramerstorfer et al. Eur J Pharmacol. .

Abstract

Benzodiazepine site agonists or inverse agonists enhance or reduce gamma-aminobutyric acid(A) (GABA(A)) receptor-mediated inhibition of neurons, respectively. Recently, it was demonstrated that the point mutation gamma 2F77I causes a drastic change in the affinity of a variety of benzodiazepine agonists or inverse agonists in receptor binding studies. Here we investigated the potency and efficacy of 10 benzodiazepine site ligands from 6 structural classes in wild-type and gamma 2F77I point mutated recombinant GABA(A) receptors composed of alpha 1 beta 3 gamma 2, alpha 2 beta 3 gamma 2, alpha 3 beta 3 gamma 2, alpha 4 beta 3 gamma 2, alpha 5 beta 3 gamma 2, and alpha 6 beta 3 gamma 2 subunits. Results indicate that the effects of the benzodiazepine site ligands zolpidem, zopiclone, Cl218872, L-655,708 and DMCM were nearly completely eliminated in all mutated receptors up to a 1 microM concentration. The effects of bretazenil, Ro15-1788 or abecarnil were eliminated in some, but not all mutated receptors, suggesting that the gamma 2F77I mutation differentially influences the actions of these ligands in different receptor subtypes. In addition, this point mutation also influences the efficacy of diazepam for enhancing GABA-induced chloride flux, suggesting that the amino acid residue gamma 2F77 might also be involved in the transduction of the effect of benzodiazepines from binding to gating. The application of these drugs in a novel mouse model is discussed.

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Figures

Fig. 1
Fig. 1. Structures of compounds used in this study.
Fig. 2
Fig. 2
Modulation of GABA EC3-currents in recombinant α1β3γ2 (A,C) and α1β3γ2F77I (B,D) receptors by (A,B) diazepam and (C,D) zolpidem. The higher GABA EC3 currents in B and D reflect a stronger expression of α1β3γ2F77 receptors.
Fig. 3
Fig. 3
Concentration–effect curves for A) and F) diazepam, B) and G) zolpidem, C) and H) zopiclone, D) and I) CL 218872 and E) and J) bretazenil on α1β3γ2 (■), α2β3γ2(▾), α3β3γ2(●), α4β3γ2(X), α5β3γ2(♦), α6β3γ2(*), α1β3γ2F77I(□), α2β3γ2F77I(▽), α3β3γ2F77I(○), α4β3γ2F77I(X), α5β3γ2F77I(◊) and α6β3γ2F77I(*) receptors. Data are normalized to a control GABA current at EC3. Data points represent means ± SEM from at least 3 oocytes derived from ≥ 2 batches.
Fig. 4
Fig. 4
Concentration–effect curves for A) and F) Ro15-1788, B) and G) L-655,708, C) and H) DMCM, D) and I) FG7142 and E) and J) abecarnil on α1β3γ2 (■), α2β3γ2(▾), α3β3γ2(•), α4β3γ2(X), α5β3γ2(♦), α6β3γ2(*), α1β3γ2F77I(□), α2β3γ2F77I(▽), α3β3γ2F77I(○), α4β3γ2F77I(X), α5β3γ2F77I(◊) and α6β3γ2F77I(*) receptors. Data are normalized to a control GABA current at EC3. Data points represent means ± SEM from at least 3 oocytes derived from ≥ 2 batches.
Fig. 5
Fig. 5
GABA dose–response curves of α1β3γ2(■), α2β3γ2(▾), α3β3γ2(●), α4β3γ2(X), α5β3γ2(♦), α6β3γ2(*), receptors (Fig. 5A) and α1β3γ2F77I(□), α2β3γ2F77I(▽), α3β3γ2F77I(○), α4β3γ2F77I(X), α5β3γ2F77I(◊), and α6β3γ2F77I(*) receptors (Fig. 5B). Data are normalized to maximum GABA current. Data points represent means ± SEM from at least 3 oocytes derived from ≥ 2 batches. The maximum GABA-induced currents were 6 ± 0.4 µA (n = 43), 4 ± 0.4 µA (n = 21), 6 ± 0.7 µA (n =14), 1 ± 0.1 µA (n = 22), 8 ± 0.9 µA (n =14) and 1 ± 0.1 µA (n = 20) for α1β3γ2, α2β3γ2, α3β3γ2, α4β3γ2, α5β3γ2 and α6β3γ2 receptors and 5 ± 0.7 µA (n = 21), 7 ± 0.9 µA (n = 22), 3 ± 0.3 µA (n =19), 2 ± 0.2 µA (n =19), 7 ± 0.7 µA (n =18) and 1 ± 0.1 µA (n = 14) for α1β3γ2F77I, α2β3γ2F77I, α3β3γ2F77I, α4β3γ2F77I, α5β3γ2F77I and α6β3γ2F77I receptors (data represent means ± SEM, number of experiments is given in parenthesis). The EC50 values were 47 ± 5 µM, 19 ± 3 µM, 79 ± 5 µM, 20 ± 5 µM, 8 ± 0.4 µM and 15 ± 2 µM for α1β3γ2, α2β3γ2, α3β3γ2, α4β3γ2, α5β3γ2 and α6β3γ2 receptors and 67 ± 6 µM, 35 ± 3 µM, 94 ± 4 µM, 15 ± 2 µM, 17 ± 3 µM and 18 ± 6 µM for α1β3γ2F77I, α2β3γ2F77I, α3β3γ2F77I, α4β3γ2F77I, α5β3γ2F77I and α6β3γ2F77I receptors. The values for the Hill coefficient were 1.3, 1.4, 1.4, 0.7, 1.3 and 0.6 for α1β3γ2, α2β3γ2, α3β3γ2, α4β3γ2, α5β3γ2 and α6β3γ2 receptors and 1.2, 0.8, 1.1, 0.8, 0.9 and 0.7 for α1β3γ2F77I, α2β3γ2F77I, α3β3γ2F77I, α4β3γ2F77I, α5β3γ2F77I and α6β3γ2F77I receptors.
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