LACTONE MODULATION OF THE GAMMA-AMINOBUTYRIC-ACID-A RECEPTOR - EVIDENCE FOR A POSITIVE MODULATORY SITE

The gamma-aminobutyric acid-A (GABA(A)) receptor complex is allosteric ally modulated by a variety of substances, some of clinical importance . Barbiturates and neurosteroids augment GABA-currents and also direct ly gate the channel. A variety of gamma-butyrolactone analogues also m odulate GABA-induced currents, with some potentiating and others inhib iting. Because several gamma-thiobutyrolactone analogues have biphasic effects on GABA currents, experiments with wild-type and picrotoxinin -insensitive GABA(A) receptors were performed to analyze whether some gamma-thiobutyrolactones interact with two distinguishable sites on th e GABA(A) receptor. beta-Ethyl-beta-methyl-y-thiobutyrolactone inhibit ed GABA-induced currents at low concentrations (0.001-1 mM), but poten tiated GABA-induced currents at higher concentrations (3-10 mM) in wil d-type alpha 1 beta 2 gamma 2-subunit containing ionophores. The relat ed alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone potentiated submax imal GABA currents in wild-type receptors at both low and high concent rations (0.1-10 mM). Mutations in the second transmembrane domain of a lpha 1, beta 2, or gamma 2 conferred picrotoxinin-insensitivity onto G ABA(A) receptor complexes. When these mutated alpha 1, beta 2, or gamm a 2 subunits were incorporated into the receptor complex, beta-ethyl-b eta-methyl-beta-thiobutyrolactone potentiated GABA currents over the e ntire concentration range (0.1-10 mM). Neither the potentiating activi ty nor the EC50 of alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone ch anged in the mutant receptors. Further studies demonstrated that the m utations 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, benzodia zepines, and steroids. Additionally, they show that the gamma-butyrola ctones probably interact at two different sites on the ionophore to pr oduce opposite effects on GABA-mediated current.