Tryptophan substitutions were made in the second transmembrane domain of the gamma-aminobutyric acid(A) (GABA(A)) receptor alpha and beta subunits and the resulting mutant receptors, containing alpha(2)(S270W) and/or beta(1)(S265W), were expressed in Xenopus oocytes. Mutation of either or both subunits resulted in receptors that exhibited enhanced sensitivity to agonist and were spontaneously active in the absence of GABA. The spontaneous activity was blocked by picrotoxin or bicuculline. The enhancement of GABA-induced currents by pentobarbital, by the neurosteroid 5alpha-pregnan-3alpha-ol-20-one, and by the benzodiazepine flunitrazepam was dramatically reduced in the mutant receptors. These results are consistent with the idea that a mutation that promotes gating behavior in a ligand-gated ion channel will also show reduced effects of all positive allosteric modulators in a generalized manner, even when these modulators act at distinct sites on the receptor.