Modulation of GABA(A) receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABA(A) receptor isoforms

Neuroactive steroids are potent, selective allosteric modulators of gamma-a minobutyric acid type A (GABA(A)) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In ord er to study the influence of subunit subtypes of the GABA(A) receptor on mo dulation of receptor function by neuroactive steroids, we expressed human r ecombinant GABA(A) receptors in Xenopus. oocytes. GABA-activated membrane c urrent, and the modulatory effects of the endogenous neurosteroid 5 alpha-p regnan-3 alpha-ol-20-one (allopregnanolone) and the synthetic steroid anest hetic 5 alpha-pregnan-3 alpha-ol-11,20-dione (alphaxalone) were measured us ing two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha 1 beta 1 gamma 2L and alpha 1 beta 2 gamma 2L receptor isoforms. In contras t, alphaxalone was much more effective as a positive allosteric modulator o n the alpha 1 beta 1 gamma 2L receptor isoform. In the absence of the gamma 2L subunit subtype, allopregnanolone had much greater efficacy, but its po tency was decreased. Allopregnanolone was much more effective on the alpha 1 beta 1 receptor isoform compared with the alpha 1 beta 2 receptor isoform . The potency for alphaxalone to potentiate the GABA response was not alter ed in the absence of the gamma 2L subunit subtype, although its efficacy wa s greatly enhanced. Both allopregnanolone and alphaxalone produced nonparal lel leftward shifts in the GABA concentration-response relationship in the absence of the gamma 2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma 2L subunit subtype was present. The 3 beta-pr egnane isomers epipregnanolone and isopregnanolone both inhibited the abili ty of allopregnanolone and alphaxalone to potentiate GABA(A) receptor funct ion. However, the degree of block produced by the 3 beta-pregnane steroid i somers was dependent on the type of receptor isoform studied and the neuroa ctive steroid tested. Isopregnanolone, the 3 beta-isomer of allopregnanolon e, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha 1 beta 2 gamma 2L receptor isoform compared with the alpha 1 beta 1 gamma 2L recepto r isoform, and also produced a greater degree of block of potentiation caus ed by allopregnanolone compared with alphaxalone. Our results support the h ypothesis that the heteromeric assembly of different GABA(A) receptor isofo rms containing different subunit subtypes results in multiple steroid recog nition sites on GABA(A) receptors, which in turn produces distinctly differ ent modulatory interactions between neuroactive steroids acting at the GABA (A) receptor. The alpha and gamma subunit subtypes may have the greatest in fluence on allopregnanolone modulation of GABA(A) receptor function, wherea s the beta and gamma subunit subtypes appear to be most important for the m odulatory effects of alphaxalone.