The influence of neuroactive steroid lipophilicity on GABA_A receptor modulation: Evidence for a low-affinity interaction

Anesthetic steroids with actions at γ-aminobutyric acid type A receptors (GABA_ARs) may access transmembrane domain binding site(s) directly from the plasma cell membrane. Accordingly, the effective concentration in lipid phase and the ability of the steroid to meet pharmacophore requirements for activity will both contribute to observed steady-state potency. Furthermore, onset and offset of receptor effects may be rate limited by lipid partitioning. Here we show that several GABA-active steroids, including naturally occurring neurosteroids, of different lipophilicity differ in kinetics and potency at GABA_ARs. The hydrophobicity ranking predicted relative potency of GABA_AR potentiation and predicted current offset kinetics. Kinetic offset differences among steroids were largely eliminated by γ-cyclodextrin, a scavenger of unbound steroid, suggesting that affinity differences among the analogues are dwarfed by the contributions of nonspecific accumulation. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-tagged fluorescent analogue of the low-lipophilicity alphaxalone (C17-NBD-alphaxalone) exhibited faster nonspecific accumulation and departitioning than those of a fluorescent analogue of the high-lipophilicity (3α,5α)-3-hydroxypregnan-20-one (C17-NBD-3α5αA). These differences were paralleled by differences in potentiation of GABA_AR function. The enantiomer of C17-NBD-3α5αA, which does not satisfy pharmacophore requirements for steroid potentiation, exhibited identical fluorescence kinetics and distribution to C17-NBD-3α5αA, but was inactive at GABA _ARs. Simple simulations supported our major findings, which suggest that neurosteroid binding affinity is low. Therefore both specific (e.g., fulfilling pharmacophore requirements) and nonspecific (e.g., lipid solubility) properties contribute to the potency and longevity of anesthetic steroid action.