Intrasubunit and Intersubunit Steroid Binding Sites Independently and Additively Mediate α1β2γ2L GABAA Receptor Potentiation by the Endogenous Neurosteroid Allopregnanolone

Prior work employing functional analysis, photolabeling, and X-ray crystallography have identified three distinct binding sites for potentiating steroids in the heteromeric GABA(A) receptor. The sites are located in the membrane-spanning domains of the receptor at the, beta-alpha subunit interface (site I) and within the alpha (site II) and beta subunits (site III). Here, we have investigated the effects of mutations to these sites on potentiation of the rat alpha 1 beta 2 gamma 2L GABA(A) receptor by the endogenous neurosteroid allopregnanolone (alpha 5 alpha P). The mutations were introduced alone or in combination to probe the additivity of effects. We show that the effects of amino acid substitutions in sites I and II are energetically additive, indicating independence of the actions of the two steroid binding sites. In site III, none of the mutations tested reduced potentiation by 3 alpha 5 alpha P, nor did a mutation in site III modify the effects of mutations in sites I or II. We infer that the binding sites for 3 alpha 5 alpha P act independently. The independence of steroid action at each site is supported by photolabeling data showing that mutations in either site I or site II selectively change steroid orientation in the mutated site without affecting labeling at the unmutated site. The findings are discussed in the context of linking energetic additivity to empirical changes in receptor function and ligand binding. SIGNIFICANCE STATEMENT Prior work has identified three distinct binding sites for potentiating steroids in the heteromeric gamma-aminobutyric acid type A receptor. This study shows that the sites act independently and additively in the presence of the steroid allopregnanolone and provide estimates of energetic contributions made by steroid binding to each site.

Automated summary

Previous work has identified three distinct binding sites for potentiating steroids in the heteromeric GABA(A) receptor, with mutations in these sites showing independent and additive effects. The findings suggest that mutations in sites I and II have energetically additive effects, while mutations in site III do not affect potentiation by the steroid. The results indicate that steroid actions at each binding site are independent.