Modulation of the human ρ1 GABAA receptor by inhibitory steroids
- 256 Downloads
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.
KeywordsGABA receptor Ion channel Neurosteroid Mutagenesis Fluorescence
This work was supported by the National Institutes of Health Grant GM47969. We thank Drs. Chuck Zorumski and Steve Mennerick for the Xenopus laevis oocytes and Dr. Joe Henry Steinbach for the many stimulating discussions and comments on the manuscript.
- Chebib M, Hinton T, Schmid KL, Brinkworth D, Qian H, Matos S, Kim HL, Abdel-Halim H, Kumar RJ, Johnston GA, Hanrahan JR (2009) Novel, potent, and selective GABAC antagonists inhibit myopia development and facilitate learning and memory. J Pharmacol Exp Ther 328:448–457PubMedCentralPubMedCrossRefGoogle Scholar