ABSTRACT
Alkyl-substituted butyrolactones have both inhibitory and stimulatory effects on GABA(A) receptors. Lactones with small alkyl substitutions at the alpha-position positively modulate the channel, whereas beta-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, alpha-benzyl-alpha-methyl-gamma-butyrolactone (alpha-BnMeGBL), on the GABA(A) receptor. R-(-)-alpha-BnMeGBL was 2-fold more potent than the S-(+)-alpha-BnMeGBL in blocking pentylenetetrazol-induced seizures in CF-1 mice. The (+)-enantiomer inhibited binding of t-butylbicyclophosporothionate with a higher affinity than the (-)-enantiomer (IC(50) of 0.68 and 1.1 mM, respectively). Whole cell patch-clamp recordings from recombinant alpha1beta2gamma2 receptors stably expressed in HEK293 cells demonstrated that both compounds stimulated GABA-activated current. The maximal stimulation was approximately 2-fold greater with (+)-alpha-BnMeGBL than that seen with (-)-alpha-BnMeGBL. Both enantiomers of alpha-BnMeGBL directly gated the GABA(A) receptor at mM concentrations, in a nonstereoselective manner. Our data demonstrate the stimulatory actions of alpha-BnMeGBL on GABA(A) receptor function display enantioselectivity and provide strong evidence for the existence of a true "lactone site" on the receptor.