The influence of the membrane on neurosteroid actions at GABA(A) receptors

Abstract

Modern views of anesthetic neurosteroid interaction with the GABA(A) receptor conceptualize steroid ligands interacting with a protein binding site on the receptor. It has generally been assumed that the steroid interaction/binding site is contained in an extracellular domain of the receptor, and that steroid interactions are of high potency, evidenced by the low aqueous ligand concentrations required to achieve potentiation of channel function. We have been considering implications of the observations that steroids are quite lipophilic and that recently identified putative steroid binding sites are in transmembrane domains of the receptor. Accordingly, we expect that both the effective plasma membrane steroid concentration and steroid pharmacophore properties will contribute to steady-state potency and to the lifetime of steroid actions following removal of free aqueous steroid. Here we review our recent studies that address the evidence that membrane partitioning and intracellular accumulation are non-specific contributors to the effects of anesthetic steroids at GABA(A) receptors. We compare and contrast the profile of anesthetic steroids with that of sulfated steroids that negatively regulate GABA(A) receptor function. These studies give rise to the view that the inherent affinity of anesthetic steroid for GABA(A) receptors is very low; low effective aqueous concentrations are accounted for by lipid partitioning. This yields a very different picture of the interaction of neurosteroids with the GABA(A) receptor than that of steroid interactions with classical intracellular steroid receptors, which exhibit inherently high affinity. These considerations have practical implications for actions of endogenous neurosteroids. Lipophilicity will tend to promote autocrine actions of neurosteroids at GABA(A) receptors within cells that synthesize neurosteroids, and lipophilic retention will limit intercellular diffusion from the source of steroid synthesis. Lipophilicity and steroid access to the receptor binding sites also must be considerations in drug design if drugs are to effectively reach the target GABA(A) receptor site.

Figure 1

The effect of γ-cyclodextrin, a steroid scavenger, on the offset times of various steroid effects. These experiments were performed on cultured postnatal hippocampal neurons grown for 8-12 days in vitro. Whole-cell recordings were performed in the presence of glutamate receptor blockers and a pipette solution containing cesium methanesulfonate. Cells were clamped at -20 mV; thus chloride flow was inward. A. Effect of γ-cyclodextrin (500 μM) on direct gating of GABA receptors (in the absence of GABA) by 300 nM 3α5αP. Horizontal bars indicate time of application of the various compounds. B. Effect on the offset of GABA receptor potentiation upon removal of steroid and wash back to GABA alone. GABA concentration was 0.5 μM. 3α5αP concentration was 300 nM, and γ-cyclodextrin concentration was 500 μM. C. Effect of 500 μM γ-cyclodextrin on antagonism offset (5 μM pregnenolone sulfate, PS). For this experiment, GABA concentration was 2 μM. The data in B and C are adapted from (Shu et al., 2007).

Figure 2

Fluorescence images of a plasma membrane marker (DiI; left panel) and 100 nM of an NBD-tagged fluorescent steroid middle panel; (Akk et al., 2005) in hippocampal neurons. Note the strong intracellular accumulation. The right panel is a merged image. Scale bar is 10 μ.

Figure 3

Various naturally occurring steroids (3α5αP and 3α5αTHDOC) and synthetic anesthetic analogues (alphaxalone and alphadolone). Estimated logP values are given from an average of 11 different algorithms (ALOGPS 2.1, http://www.vcclab.org/lab/alogps/; ChemAxon, http://www.chemaxon.com/marvin/sketch/index.jsp) demonstrating lipophilicity range of steroids with GABA_A receptor actions.