Eaton Megan M, Lim You Bin, Covey Douglas F, Akk Gustav
Department of Anesthesiology, Washington University School of Medicine, Campus Box 8054, 660 S. Euclid Ave, St. Louis, MO, 63110, USA.
Psychopharmacology (Berl). 2014 Sep;231(17):3467-78. doi: 10.1007/s00213-013-3379-z. Epub 2013 Dec 7.
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.
We employed electrophysiology and voltage-clamp fluorometry to compare the actions of several neuroactive steroids and analogues on the human ρ1 GABAA receptor.
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 17β-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 3α5βP but was without effect on modulation by (3α,5β)-3-hydroxypregnan-20-one sulfate or 17β-estradiol. The effect of 3α5βP 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 5β-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 ρ1 GABAA receptor: 5β-reduced uncharged steroids, sulfated and carboxylated steroids, and 17β-estradiol. Further division among 5β-reduced uncharged steroids was based on substituent at position C17.
ρ1 GABAA受体调节剂可能对视觉、睡眠和认知障碍的治疗有用。神经活性甾体及其类似物已被证明可调节ρ1受体功能，但分子机制尚不清楚。
我们采用电生理学和电压钳荧光法比较几种神经活性甾体及其类似物对人ρ1 GABAA受体的作用。
结果证实，P294S和T298F突变影响甾体的调节作用。P294S突变消除了(3α,5β)-3-羟基孕烷-20-酮(3α5βP)的抑制作用，而T298F突变消除了17β-雌二醇的抑制作用。电压钳荧光法表明，C3上带电荷基团或C17上取代基性质不同的甾体对细胞外结构域中GABA引起的荧光变化有独特的修饰作用。I307Q突变逆转了3α5βP的抑制作用，但对(3α,5β)-3-羟基孕烷-20-酮硫酸盐或17β-雌二醇的调节作用无影响。3α5βP对Y241C处产生的荧光变化的影响取决于甾体是作为抑制剂还是增强剂起作用。此外，该作用仅限于C17上含有乙酰基的不带电荷的5β-还原甾体。
数据表明，甾体及其类似物在这些药物引起的构象变化以及对突变效应的敏感性方面存在差异。根据它们对ρ1 GABAA受体的作用，甾体及其类似物可初步分为三大类：5β-还原不带电荷的甾体、硫酸化和羧化甾体以及17β-雌二醇。5β-还原不带电荷的甾体之间的进一步分类基于C17位的取代基。
