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

We have examined alpha 1 beta 2 gamma 2L GABA(A) receptor modulation by the endogenous steroids allopregnanolone (3 alpha 5 alpha P), pregnenolone sulfate, and beta-estradiol in the absence and presence of ethanol. Coapplication of 0.1 to 1% (17-170 mM) ethanol influenced receptor modulation by 3 alpha 5 alpha P but not that by pregnenolone sulfate or beta-estradiol. One of the three kinetic effects evident in channel potentiation by 3 alpha 5 alpha 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 alpha 5 alpha P to prolong OT3 also affected OT3 prolongation in the presence of ethanol. A mutation to the gamma 2 subunit, which reduces the ability of 3 alpha 5 alpha P to prolong OT3, also reduces the interaction between ethanol and 3 alpha 5 alpha P. And the presence of the competitive steroid antagonist (3 alpha,5 alpha)-17-phenylandrost-16-en-3-ol (17-PA) diminishes the positive interaction between ethanol and 3 alpha 5 alpha 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 alpha 5 alpha P for the site. Tadpole behavioral assays showed that the presence of 3 alpha 5 alpha 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 alpha 5 alpha P.