EFFECTS OF STEROIDS ON GAMMA-AMINOBUTYRIC-ACID RECEPTORS EXPRESSED IN XENOPUS OOCYTES BY POLY(A)+ RNA FROM MAMMALIAN BRAIN AND RETINA

Electrical recordings were made in Xenopus oocytes to study the modulatory effects of steroids on gamma-aminobutyric acid (GABA) receptors expressed by RNA from mammalian brain and retina. GABA responses expressed by rat cerebral cortex poly(A)+ RNA were bicuculline-sensitive Cl- currents mediated by GABA(A) receptors. GABA responses expressed by bovine retina poly(A)+ RNA also were Cl- currents but were composed of two pharmacologically distinct components, one mediated by GABA(A) receptors and the other by GABA receptors with novel properties, which were resistant to bicuculline but were not activated by R(+)-baclofen, a selective agonist of GABA(B) receptors. As reported in neurons and in other expression systems, GABA(A) responses expressed in oocytes by cerebral cortex RNA were strongly and stereospecifically potentiated by 5-alpha-pregnan-3-alpha-ol-20-one (3-alpha-OH-DHP) and 5-alpha-pregnan-3-alpha,21-diol-20-one (THDOC). Threshold levels of potentiation were detectable using 1-2 nM steroid, and at concentrations of 50 and 500 nM 3-alpha-OH-DHP shifted the EC50 of cortex GABA(A) responses from a control value of 92 +/- 20-mu-M GABA to 40 +/- 4.3-mu-M and 13 +/- 1.8-mu-M, respectively. However, even at concentrations as high as 50-mu-M, 3-alpha-OH-DHP did not itself eleicit appreciable membrane current responses through direct activation of the cortex GABA(A) receptors. In addition to potentiation, 3-alpha-OH-DHP and THDOC caused pronounced increases in the rate of desensitization of GABA(A) responses expressed by cortex RNA. Decay time courses of currents elicited by 1 mM GABA (90-95% of the maximum response) were fitted by the sum of two exponentials. Under control conditions, the time constant of the fast component was 4.4 +/- 0.6 sec and the slow component, 22.5 +/- 4.8 sec. 3-alpha-OH-DHP at 500 nM and 5-mu-M reduced the time constant of the fast component by 52 +/- 7% and 84 +/- 5%, respectively, but showed little effect on the slow component. Unlike the potentiation effect, actions of pregnanolones on desensitization did not show stringent stereoselectivity, and 5-mu-M 5-beta-pregnan-3-beta-ol-20-one (3-beta-OH-DHP) reduced the time constant of the fast component by 59 +/- 11%. Modulatory effects of 5-pregnen-3-beta-ol-20-one sulfate (pregnenolone-SO4) and progesterone on GABA(A) receptors expressed by rat cortex RNA were predominantly inhibitory, even though at concentrations between 2 and 20 nM pregnenolone-SO4 did cause some modest potentiation of currents elicited by 1-10-mu-M GABA. Pregnenolone-SO4 at 500 nM and 5-mu-M blocked maximum GABA responses by 38 +/- 2% and 76 +/- 8%, respectively, and the same concentrations of progesterone blocked maximum currents by 14 +/- 2% and 58 +/- 5%. For both steroids, suppression of responses was associated with only small changes in EC50, indicating that inhibition was substantially non-competitive. Time courses of GABA(A) responses with pregenolone-SO4 and progesterone did, however, appear to be different, which in turn suggested that the mechanisms underlying inhibition were not identical. Modulatory effects of steroids on GABA(A) receptors expressed by retina RNA were qualitatively indistinguishable from effects on cortex GABA(A) receptors. GABA(A) responses expressed by retina RNA were stereoselectively potentiated by 3-alpha-OH-DHP and noncompetitively inhibited by pregnenolone-SO4 and progesterone. Furthermore, 3-alpha-OH-DHP, THDOC, and 3-beta-OH-DHP also caused clear increases in the rate of response desensitization. In striking contrast, bicuculline-resistant GABA responses expressed by retina RNA were essentially unaffected by any of the steroids tested. These experiments indicate that GABA(A) receptors expressed in oocytes by cortex or retina RNA are modulated by steroids in ways similar to those of GABA(A) receptors studied in situ. Conversely, the novel bicuculline-resistant GABA receptors expressed by retina RNA do not appear to be functionally modulated by steroids.