Modulation of the human ρ1 GABAA receptor by inhibitory steroids

Modulators of the rho 1 GABA(A) receptor may be useful in the treatment of visual, sleep, and cognitive disorders. Neuroactive steroids and analogues have been shown to modulate rho 1 receptor function, but the molecular mechanisms are poorly understood. We employed electrophysiology and voltage-clamp fluorometry to compare the actions of several neuroactive steroids and analogues on the human rho 1 GABA(A) receptor. Results confirmed that P294S and T298F mutations affect modulation by steroids. The P294S mutation abolished inhibition by (3 alpha,5 beta)-3-hydroxypregnan-20-one (3 alpha 5 beta P) while the T298F mutation eliminated inhibition by 17 beta-estradiol. Voltage-clamp fluorometry demonstrated that steroids differing in the presence of a charged group on C3 or nature of substituent on C17 uniquely modified fluorescence changes elicited by GABA in the extracellular domain. The I307Q mutation reversed the inhibitory effect of 3 alpha 5 beta P but was without effect on modulation by (3 alpha,5 beta)-3-hydroxypregnan-20-one sulfate or 17 beta-estradiol. The effect of 3 alpha 5 beta P on the fluorescence change generated at Y241C was dependent on whether the steroid acted as an inhibitor or a potentiator. Further, the effect was limited to uncharged 5 beta-reduced steroids containing an acetyl group on C17. The data demonstrate that steroids and analogues differ with respect to conformational changes elicited by these drugs as well as sensitivity to the effects of mutations. Steroids and analogues could be provisionally divided into three major groups based on their actions on the rho 1 GABA(A) receptor: 5 beta-reduced uncharged steroids, sulfated and carboxylated steroids, and 17 beta-estradiol. Further division among 5 beta-reduced uncharged steroids was based on substituent at position C17.