Neurosteroid Access to the GABA_AReceptor

Abstract

GABA A receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABA A receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABA A receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.

Mechanisms of neurosteroid interactions with GABAA receptors

“…These results are complemented by results in which we found that potentiation of inside-out patches is robust when steroid is applied directly to the inner membrane leaflet (Fig. 3G), and we have synthesized a membrane-impermeant steroid with an ionically charged Alexa group substitution at C17 that only works from the inside of the membrane (Akk et al, 2005).…”

“…4 and Akk et al, 2005). We synthesized a fluorescent analogue of 3α5αP that retained activity at GABA A receptors and found that it accumulated in lipophilic regions of the cell, including plasma membrane and lipophilic intracellular compartments (Akk et al, 2005). These imaging results complement the cell-attached patch recording results and suggest the plausibility of membrane and/or intracellular access to steroid receptor sites.…”

“…With this rationale in mind, we developed tests of whether steroids access their putative site by typical aqueous access to an extracellular binding site, or whether membrane diffusion might be more important (Akk et al, 2005). A first approach was to examine behavior of individual GABA-gated channels that were either directly exposed to aqueous steroid or sealed off from direct access.…”

“…4 and Akk et al, 2005). We synthesized a fluorescent analogue of 3α5αP that retained activity at GABA A receptors and found that it accumulated in lipophilic regions of the cell, including plasma membrane and lipophilic intracellular compartments (Akk et al, 2005).…”

Neurosteroid regulation of central nervous system development

“…The data also suggest that allopregnanolone synthesized in GABA-ergic neurons will be able to act at GABA A receptors postsynaptically or extrasynaptically (Belelli and Lambert, 2005). In glutamatergic neurons that express both 5α reductase and 3α HSD and that presumably synthesize allopregnanolone, the data suggest that allopregnanolone probably works in a paracrine fashion at GABA A receptors located on dendrites or cell bodies of those glutamatergic neurons, with or without secretion into the extracellular space, through plasma membrane lateral diffusion or through intracellular routes (Akk et al, 2005). Neurosteroid modulation of GABA-mediated currents involves G-protein and protein kinase activation Fancsik et al, 2000;Harney et al, 2003;Hodge et al, 2002;Vicini et al, 2002;Wegner et al, 2006).…”