Multiple modulatory effects of the neuroactive steroid pregnanolone on GABA(A) receptor in frog pituitary melanotrophs

1. The effects of the neuroactive steroid pregnanolone (5 beta-pregnan-3 alpha-ol-20-one) on the electrical response to GABA were investigated in cultured frog pituitary melanotrophs using the patch-clamp technique. 2. Low concentrations of pregnanolone (0.01-1 mu M) in the extracellular solution enhanced the current evoked by submaximal concentrations of GABA(A) receptor agonists and prolonged the GABA-induced inhibition of the spontaneous action potentials in a dose-dependent manner. 3. Pregnanolone augmented the opening probability of the single GABA-activated channels but did not modify the conductance levels. 4. Pregnanolone (1 mu M) shifted the GABA dose-response curve towards the low GABA concentrations, reducing the EC50 from 4.2 to 1.8 mu M. 5. Internal cell dialysis with pregnanolone (1 or 1.0 mu M) did not alter the GABA-evoked current. 6. Pregnanolone accelerated the desensitization of both the current and conductance increases caused by GABA. 7. High concentrations of pregnanolone (30 mu M) markedly and reversibly diminished the current evoked by 10 mu M GABA. 8. At high concentrations (10-30 mu M), pregnanolone induced an outward current which reversed at the chloride equilibrium potential. 9. It is concluded that, in frog pituitary melanotrophs, pregnanolone exerts a dual inverse modulation and a direct activation of the GABA(A) receptor-channel depending on the concentrations of both GABA and steroid. Pregnanolone acts on an extracellular site on the GABA(A) receptor inducing conformational changes of the receptor-channel complex, resulting in a desensitized less-conducting state.