Abstract

Alkyl-substituted butyrolactones have both inhibitory and stimulatory effects on GABA_A receptors. Lactones with small alkyl substitutions at the α-position positively modulate the channel, whereas β-substituted lactones tend to inhibit the GABA_A receptor. These compounds mediate inhibition through the picrotoxin site of the receptor. A distinct binding site that mediates the stimulatory actions of lactones is presumed to exist, although no definitive evidence to support this claim exists. In the present study, we used in vivo and in vitro assays to evaluate the effects of the enantiomers of a novel lactone, α-benzyl-α-methyl-γ-butyrolactone (α-BnMeGBL), on the GABA_A receptor. R-(-)-α-BnMeGBL was 2-fold more potent than the S-(+)-α-BnMeGBL in blocking pentylenetetrazol-induced seizures in CF-1 mice. The (+)-enantiomer inhibited binding of t-butylbicyclophosporothionate with a higher affinity than the (-)-enantiomer (IC₅₀ of 0.68 and 1.1 mM, respectively). Whole cell patch-clamp recordings from recombinant α1β2γ2 receptors stably expressed in HEK293 cells demonstrated that both compounds stimulated GABA-activated current. The maximal stimulation was approximately 2-fold greater with (+)-α-BnMeGBL than that seen with (-)-α-BnMeGBL. Both enantiomers of α-BnMeGBL directly gated the GABA_A receptor at mM concentrations, in a nonstereoselective manner. Our data demonstrate the stimulatory actions of α-BnMeGBL on GABA_A receptor function display enantioselectivity and provide strong evidence for the existence of a true “lactone site” on the receptor.