Lactone Modulation of the γ-Aminobutyric Acid A Receptor: Evidence for a Positive Modulatory Site

Abstract

The γ-aminobutyric acid-A (GABA_A) receptor complex is allosterically modulated by a variety of substances, some of clinical importance. Barbiturates and neurosteroids augment GABA-currents and also directly gate the channel. A variety of γ-butyrolactone analogues also modulate GABA-induced currents, with some potentiating and others inhibiting. Because several γ-thiobutyrolactone analogues have biphasic effects on GABA currents, experiments with wild-type and picrotoxinin-insensitive GABA_A receptors were performed to analyze whether some γ-thiobutyrolactones interact with two distinguishable sites on the GABA_A receptor. β-Ethyl-β-methyl-γ-thiobutyrolactone inhibited GABA-induced currents at low concentrations (0.001–1 mm), but potentiated GABA-induced currents at higher concentrations (3–10 mm) in wild-type α1β2γ2-subunit containing ionophores. The related α-ethyl-α-methyl-γ-thiobutyrolactone potentiated submaximal GABA currents in wild-type receptors at both low and high concentrations (0.1–10 mm). Mutations in the second transmembrane domain of α1, β2, or γ2 conferred picrotoxinin-insensitivity onto GABA_A receptor complexes. When these mutated α1, β2, or γ2 subunits were incorporated into the receptor complex, β-ethyl-β-methyl-γ-thiobutyrolactone potentiated GABA currents over the entire concentration range (0.1–10 mm). Neither the potentiating activity nor the EC₅₀ of α-ethyl-α-methyl-γ-thiobutyrolactone changed in the mutant receptors. Further studies demonstrated that the mutations did not affect the EC₅₀ of chlordiazepoxide or phenobarbital. These and our earlier results identify a modulatory site on the GABA_A receptor distinct from that interacting with barbiturates, benzodiazepines, and steroids. Additionally, they show that the γ-butyrolactones probably interact at two different sites on the ionophore to produce opposite effects on GABA-mediated current.