Ethanol modulates the interaction of the endogenous neurosteroid allopregnanolone with the α1β2γ2L GABA_A receptor

We have examined α1β2γ2L GABA_A receptor modulation by the endogenous steroids allopregnanolone (3α5αP), pregnenolone sulfate, and β-estradiol in the absence and presence of ethanol. Coapplication of 0.1 to 1% (17-170 mM) ethanol influenced receptor modulation by 3α5αP but not that by pregnenolone sulfate or β-estradiol. One of the three kinetic effects evident in channel potentiation by 3α5αP, 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 3α5αP to prolong OT3 also affected OT3 prolongation in the presence of ethanol. A mutation to the γ2 subunit, which reduces the ability of 3α5αP to prolong OT3, also reduces the interaction between ethanol and 3α5αP. And the presence of the competitive steroid antagonist (3α,5α)-17-phenylandrost-16-en-3-ol (17-PA) diminishes the positive interaction between ethanol and 3α5αP 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 3α5αP for the site. Tadpole behavioral assays showed that the presence of 3α5αP 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 3α5αP.