Modulation of the human ρ 1G ABA A receptor by inhibitory steroids

Rationale Modulators of the ρ1 GABAA receptor may be useful in the treatment of visual, sleep, and cognitive disorders. Neuroactive steroids and analogues have been shown to modulate ρ1 receptor function, but the molecular mechanisms are poorly understood. Objectives We employed electrophysiology and voltageclamp fluorometry to compare the actions of several neuroactive steroids and analogues on the human ρ1 GABAA receptor. Results Results confirmed that P294S and T298F mutations affect modulation by steroids. The P294S mutation abolished inhibition by (3α,5β)-3-hydroxypregnan-20-one (3α5βP) while the T298F mutation eliminated inhibition by 17βestradiol. 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 3α5β Pb ut was without effect on modulation by (3α,5β)-3hydroxypregnan-20-one sulfate or 17β-estradiol. The effect of 3α5βP on the fluorescence change generated at Y241C wasdependentonwhetherthesteroidactedasaninhibitorora potentiator. Further, the effect was limited to uncharged 5βreduced steroids containing an acetyl group on C17. Conclusions The data demonstrate that steroids and analogues differ with respect to conformational changes elicited bythesedrugsaswellassensitivitytothe effectsofmutations. Steroids and analogues could be provisionally divided into three major groups based on their actions on the ρ 1G ABAA receptor: 5β-reduced uncharged steroids, sulfatedand carboxylated steroids, and 17β-estradiol. Further division among 5β-reduced uncharged steroids was based on substituent at position C17.