Multiple structural features of steroids mediate subtype-selective effects on human α4β3δ GABAA receptors

Neurosteroids have been shown to mediate some of their physiological effects via a modulatory site on type A inhibitory γ-aminobutyric acid (GABAA) receptors. In particular, recent evidence has implicated selective potentiation of the δ subunit of GABAA receptors as an important mediator of in vitro and in vivo neurosteroid activity. However, this has been demonstrated for only a very small number of steroids, so both the generality of this finding, and the structural features of steroids which mediate functional δ-selectivity, are unclear. We have used a potentiometric assay based on fluorescence resonance energy transfer to measure GABA-activated responses in L(tk−) cells stably transfected with human GABAA receptor α4β3δ and α4β3γ2 receptor subtypes. A set of 28 steroids were evaluated on these subtypes to characterise their functional potency and efficacy in modulating GABA responses. For most compounds there was a clear separation of their efficacy profiles between the receptor subtypes, with a substantially larger maximal response at the α4β3δ receptor. 5β-Pregnan-3β-ol-20-one, 5β-pregnane-3α,20β-diol and 5β-pregnane-3α,17α-diol-11,20-dione showed particularly high efficacy for α4β3δ. No compounds were identified that simply inhibited responses at δ-containing receptors. However, 5β-pregnane-3α,17α,20β-triol, prednisolone 21-acetate, 4-pregnene-17α,20α-diol-3-one-20-acetate, 4-pregnen-20α-ol-3-one, and 5β-pregnane-3α,17α,21-triol-20-one inhibited, though did not abolish, GABA responses at the α4β3γ2 subtype, while evoking modest-amplitude potentiation of α4β3δ responses. Molecular modelling on this compound series using principal components analysis indicates that several structural features of steroids underlie their relative functional selectivity for potentiation of δ-containing GABAA receptors.