Biphasic modulation of GABA(A) receptor binding by steroids suggests functional correlates

Neuroactive steroids and other positive modulators of GABA, receptors showe d regional variation in both the efficacy and potency for modulation of [S- 35]TBPS binding to rat brain membrane homogenates, with biphasic concentrat ion-dependence. GABA present in the binding assays prevented the enhancemen t phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GA BAA receptors, expressed in insect cell line Sf9 using baculovirus, enhance ment by steroids of [S-35]TBPS binding was sensitive to the presence of the gamma 2 subunit and the nature of the a subunit (alpha 1 beta 2 gamma 2S > alpha 1 beta 2, alpha 6 beta 2, alpha 6 beta 2 gamma 2S, and alpha 6 beta 2 delta). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are con sistent with a three-state model in which the receptor mono-liganded by GAB A or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for T BPS.