Ethanol Modulates the Interaction of the Endogenous Neurosteroid Allopregnanolone with the α1β2γ2L GABAA Receptor

Abstract

We have examined ␣1␤2␥2L GABA A receptor modulation by the endogenous steroids allopregnanolone (3␣5␣P), pregnenolone sulfate, and ␤-estradiol in the absence and presence of ethanol. Coapplication of 0.1 to 1% (17-170 mM) ethanol influenced receptor modulation by 3␣5␣P but not that by pregnenolone sulfate or ␤-estradiol. One of the three kinetic effects evident in channel potentiation by 3␣5␣P, prolongation of the longest-lived open time component (OT3), was affected by ethanol with the midpoint of its dose-response curve moved to lower steroid concentrations by 2 orders of magnitude without significantly affecting the maximal effect. Manipulations designed to affect the ability of 3␣5␣P to prolong OT3 also affected OT3 prolongation in the presence of ethanol. A mutation to the ␥2 subunit, which reduces the ability of 3␣5␣P to prolong OT3, also reduces the interaction between ethanol and 3␣5␣P. And the presence of the competitive steroid antagonist (3␣,5␣)-17-phenylandrost-16-en-3-ol (17-PA) diminishes the positive interaction between ethanol and 3␣5␣P on the GABA A receptor. Together, the findings suggest that steroid interactions with the classic steroid binding site underlie the effect seen in the presence of ethanol, and that ethanol acts by increasing the affinity of 3␣5␣P for the site. Tadpole behavioral assays showed that the presence of 3␣5␣P at a concentration ineffective at causing changes in tadpole behavior shifted the ethanol dose-response curve for loss of righting reflex to lower concentrations and that this effect was neutralized by coapplication of 17-PA with 3␣5␣P.From a pharmacologist's point of view, the GABA A receptor presents a fascinating case, because the receptor can be modulated by a wide collection of structurally unrelated drugs, most with their own individual binding sites. The GABA A receptor constitutes the dominant fast inhibitory force in the central nervous system. It is unsurprising that compounds that potentiate receptor function have anesthetic or anticonvulsant properties, and conversely, that inhibitors of the GABA A receptor can act as cognitive enhancers or elicit seizures. The clinical implications have undoubtedly fueled the interest in receptor function resulting in rapid progress in recent years in the identification of interaction sites and mechanisms of action for the modulators of the GABA A receptor.Neuroactive steroids are among the best-described GABA A receptor modulators. The ability of steroids to modify GABA A receptor activity has been known since the 1980s (Harrison and Simmonds, 1984); since then, significant progress has been made in terms of describing the kinetic and structural mechanisms (e.g., Puia et al., 1990;Akk et al., 2004; Hosie et al., 2005) as well as understanding the physiological significance of steroid modulation (for reviews see, Baulieu, 1998;Belelli and Lambert, 2005). Exposure to most steroids has a major effect on gating or desensitization of the receptorchannel. Potentiating steroids (e.g., 3␣5␣P) enhance the channel open...

A new meaning for “Gin & Tonic”: tonic inhibition as the target for ethanol action in the brain

“…A recent report highlighted that effect of the neurosteroid allopregnanolone on prolonging the long open-times of single α1β2γ2L GABA A R channels expressed in HEK 293 cells activated by 50 μM GABA was shifted to 100-fold lower neurosteroid concentrations in the presence of 17 mM ethanol (Akk et al, 2007). However, this effect was not evident in the whole-cell currents evoked by GABA, possibly because of the relatively small contribution of the long openings to the total current.…”

Mechanisms of neurosteroid interactions with GABAA receptors

“…A recent study showed a similar interaction between an endogenous neurosteroid, 3α5αP, and ethanol (Akk et al, 2007). Coapplication of the two drugs, at concentrations ineffective individually, resulted in a significant increase in the duration of OT3 without affecting the relative frequency of OT3 or the channel closing rate.…”

“…First, there is an indirect effect, where the application of ethanol modifies the level of some endogenous GABA A receptor modulator without affecting the ability of the receptor to respond to the stimulus (Barbaccia et al, 1999;Morrow et al, 2001;O'Dell et al, 2004;Sanna et al, 2004;VanDoren et al, 2000). Second is a direct interaction, where exposure to ethanol modifies the ability of the receptor to respond to a stimulus, which otherwise would have been ineffective at modulating receptor function (Akk et al, 2007;Murayama et al, 2006). VanDoren et al (2000) showed that administration of ethanol was followed by an increase in the levels of 3α5αP in the rodent cerebral cortex.…”

The role of GABAA receptors in the development of alcoholism

“…These findings have not, however, been replicated in other labs (Borghese and Harris, 2007;Korpi et al, 2007). In addition, it is currently a subject of debate as to whether low concentrations of alcohol affect only α 4 , α 6 and δ subunits (Lovinger and Homanics, 2007), For example, it has been shown that co-application of low concentrations of ethanol (17mM) influences channel potentiation of at least the α 1 β 2 γ 2 receptor by neurosteroids (Akk et al, 2007). Moreover, α 1 δ subunit assemblies that are highly sensitive to low concentrations of ethanol and mediate tonic inhibitory currents have recently been detected in hippocampal interneurons (Glykys et al, 2007).…”