Benzodiazepines modulate GABA_A receptors by reducing a gamma-subunit-mediated inhibition of GABA sensitivity

Background

Heterologous expression of α₁, β₂ and γ_2S(γ₁) subunits produces a mixed population of GABA_A receptors containing α₁β₂ or α₁β₂γ_2S(γ₁) subunits. GABA sensitivity (lower in receptors containing γ₁ or γ_2S subunits) and the potentiation of GABA-activated chloride currents (I_GABA) by benzodiazepines (BZDs) are dependent on γ_2S(γ₁) incorporation [1]. A variable γ subunit incorporation may affect the estimation of I_GABA potentiation by BZDs. We propose an approach for estimation of BZD efficiency that accounts for a mixed population of α₁β₂ and α₁β₂γ_2S(γ₁) receptors.

Methods

We investigated the relation between GABA sensitivity (EC₅₀) and BZD modulation by analyzing triazolam-, clotiazepam- and midazolam-induced potentiation of I_GABA in Xenopus oocytes under two-microelectrode voltage clamp.

Results

Plotting EC₅₀ versus BZD-induced shifts of GABA concentration-response curves (ΔEC₅₀(BZD)) of oocytes injected with different amounts of α₁, β₂ and γ_2S(γ₁) cRNA (1:1:1-1:1:10) revealed a linear regression between γ_2S(γ₁)-mediated reduction of GABA sensitivity (EC₅₀) and ΔEC₅₀(BZD). The slope factors of the regression were always higher for oocytes expressing α₁β₂γ₁ subunit receptors (triazolam: 1.8 ± 0.1; clotiazepam: 1.6 ± 0.1; midazolam: 2.3 ± 0.2) than for oocytes expressing α₁β₂γ_2S receptors (triazolam: 1.4 ± 0.1; clotiazepam: 1.4 ± 0.1; midazolam: 1.3 ± 0.1). Mutant GABA_A receptors (α₁β₂-R207Cγ_2S) with lower GABA sensitivity showed higher drug efficiencies (slope factors: triazolam: 1.1 ± 0.1; clotiazepam: 1.1 ± 0.1; midazolam: 1.2 ± 0.1) whereas higher GABA sensitivity of α₁-L263Sβ₂γ_2S mutant receptors was associated with lower efficiency (slope factor: clotiazepam: 1.7 ± 0.1).

Conclusion

Regression analysis enabled the estimation of BZD efficiency when variable mixtures of α₁β₂ and α₁β₂γ_2S(γ₁) receptors are expressed and provided new insights into the γ_2S(γ₁) dependency of BZD action. The method for determining the slope of the regression line also allowed the determination of the percentage of α₁β₂γ_2S receptors. Assuming a twofold difference in the single channel conductance of α₁β₂ and α₁β₂γ_2S receptors, at 70% current ratio of γ_2S-containig receptors our mathematical model predicts only about 50% of γ_2S subunit incorporation. Our data suggest that BZDs reduce a γ-subunit-mediated inhibition of GABA sensitivity.