High affinity, heterogeneous displacement of [3H]EBOB binding to cerebellar GABA A receptors by neurosteroids and GABA agonists

Abstract

Heterogeneous binding interactions of cerebellar GABA(A) receptors were investigated with GABA agonists and neurosteroids. GABA(A) receptors of rat cerebellum were labelled with [(3)H]ethynylbicycloorthobenzoate (EBOB), a convulsant radioligand. Saturation analysis revealed a homogenous, nanomolar population of [(3)H]EBOB binding. Both GABA and 5alpha-tetrahydrodeoxycorticosterone (5alpha-THDOC) displaced [(3)H]EBOB binding heterogeneously, with nanomolar and micromolar potencies. The nanomolar phase of displacement by GABA was selectively abolished by 100 microM furosemide. Physiological concentrations of allopregnanolone (8 nM) and 5alpha-THDOC (20 nM) increased the displacing effects of nanomolar GABA. GABA (0.3 microM ) and 5alpha-THDOC (0.3 microM ) potentiated the micromolar population of displacement by the other. Taurine inhibited [(3)H]EBOB binding also heterogeneously, with micromolar and millimolar potencies, and 0.3 microM 5alpha-THDOC potentiated this inhibition. 5beta-THDOC did not affect [(3)H]EBOB binding significantly but in 1 microM it antagonised selectively the nanomolar displacement by 5alpha-THDOC. [(3)H]EBOB binding to hippocampal GABA(A) receptors was inhibited by GABA and allopregnanolone with low (micromolar) potencies and with slope values higher than unity referring to allosteric interaction. High affinity displacement of cerebellar [(3)H]EBOB binding by GABA agonists and neurosteroids can be associated with constitutively open alpha(6)betadelta GABA(A) receptors, tonic GABAergic inhibitory neurotransmission and its modulation by physiological concentrations of neurosteroids.