Insight into the mechanism of action of neuroactive steroids.

Rho(1) receptor-channels (rho(1)Rs) are GABA-gated chloride channels that exhibit slow kinetics, little desensitization, and inert pharmacology to most anesthetics, except for neuroactive steroids (NSs). NSs differentially modulate rho(1)Rs dependent on the steric arrangement of the hydrogen atom at the fifth carbon position. In particular, the NS allotetrahydrodeoxycorticosterone (5alpha-THDOC) potentiates, whereas 5beta-pregnane-3alpha-ol-20-one (pregnanolone) and 5beta-dihydroprogesterone (5beta-DHP) inhibit rho(1) GABA currents. Here, we used Xenopus laevis oocytes expressing rho(1)Rs as a model system to study the mechanism of NS modulation. The second transmembrane residue, Ile307, was mutated to 16 amino acids. Subsequent testing of these mutants with 5alpha- and 5beta-NSs, at equivalent GABA activity, showed the following paradigm. For 5beta-DHP, Ile307 mutation either altered the degree of inhibition or entirely reversed the direction of modulation, rendering 5beta-DHP a potentiator. Dependent on the mutation, pregnanolone remained an inhibitor, transformed into a potentiator, or converted to inhibitor and potentiator based on concentration. The extent of mode reversal for both 5beta compounds showed a correlation with the side-chain hydrophilicity of the 307 residue. In contrast, Ile307 substitutions did not alter the direction of modulation for 5alpha-THDOC but caused a significant increase in the level of potentiation. Paradoxical to their impact on the mode and/or the degree of modulation, none of the mutations altered the concentration range producing the response significantly for any of the above NSs. Moreover, preincubation of Ile307 mutants with 5alpha or 5beta alone produced an equivalent effect on the activation time course. Based on the above data, a universal model is presented wherein anesthetic compounds like NSs can potentiate or inhibit the activity of ligand-gated ion channels distinct from interaction with alternative binding sites.