Neuroactive Steroids and Human Recombinant 1 GABAC Receptors

The -aminobutyric acid type C (GABAC) receptor is structurally related to the GABAA receptors, yet quite distinct physiologically and pharmacologically. Neuroactive steroids are known to be potent and efficacious modulators of the GABAA receptor, but they are less well characterized in their actions on the GABAC receptor. We have examined the actions of neuroactive steroids and analogs on 1 (GABAC) receptors expressed in Xenopus laevis oocytes, with two goals in mind. First, we tested a larger number of endogenous steroids, to determine whether particularly potent steroids could be found. Second, we examined the structure-activity relationship for steroid actions, and some mechanistic features, to determine the possible numbers of steroid binding sites and mechanisms of action. In total, 41 compounds were examined. Estradiols are inhibitors, essentially equipotent with picrotoxinin. No endogenous steroid tested was highly efficacious at potentiation. The results of the structure-activity studies and the effects of two mutations to the second transmembrane region of the 1 GABAC receptor indicate that there are several mechanisms by which steroids can inhibit the receptor. Surprisingly, estradiol shares some features with picrotoxin. Inhibition by negatively charged compounds was not sensitive to membrane potential, and inhibition by all compounds tested was reduced at higher concentrations of GABA. The data indicate that the binding sites mediating potentiation and inhibition differ from each other and that there are several (three or more) mechanisms for producing inhibition. The GABAC receptor was first characterized as a bicuculline-insensitive, baclofen-insensitive GABA-activated conductance (Johnston, 1996). Molecular studies identified three subunits, 1– 3, which combine to form pentameric receptors that have properties similar to GABAC receptors in native cells (Enz and Cutting, 1998). Although the GABAC and GABAA receptors show molecular similarities, they are quite distinct in terms of their cellular localizations, probably physiological roles, and pharmacological properties (Johnston, 1996; Bormann, 2000; Johnston et al., 2003; Lukasiewicz et al., 2004). Neuroactive steroids are known to be the most potent and efficacious endogenous modulators of the GABAA receptor (Belelli et al., 2006), but their activities on GABAC receptors have been less well characterized. Initial studies of receptors expressed in Xenopus oocytes after injections of retinal mRNA found that the bicuculline-insensitive responses to GABA were insensitive to both potentiating and inhibiting neurosteroids (Woodward et al., 1992). However, a subsequent study found that the apparent insensitivity resulted from the fact that relatively high concentrations of GABA were used to elicit responses; when low concentrations of GABA were used, both potentiation and inhibition could be shown (Morris et al., 1999). However, the effects required higher steroid concentrations than those necessary for modulation of the GABAA receptor. We have undertaken a further study of the actions of steroids on the 1 GABAC receptor, to examine a broader range of endogenous steroids and to better define the structure-activity requirements for steroid actions. There were two primary motivations for these studies. The first motivation was to determine whether some endogenous steroids have higher potency or efficacy than those examined to date. Because steroids are so effective in actions on the GABAA receptor, it seemed possible that a particular steroid might demonstrate a physiologically relevant selectivity for the GABAC receptor. The GABAC receptor plays a less well defined role in the function of the nervous system (Zhang et al., 2001). It has a clear role in the retina (Lukasiewicz et al., 2004), and studies of genetically modified mice suggest that it may play a role in This work was supported by National Institutes of Health Grant P01 GM 47969 (to D.F.C. and J.H.S.). J.H.S. is the Shelden Professor of Anesthesiology. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.127365. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: DMSO, dimethyl sulfoxide; TM, transmembrane. 0022-3565/07/3231-236–247$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 323, No. 1 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 127365/3256927 JPET 323:236–247, 2007 Printed in U.S.A. 236 http://jpet.aspetjournals.org/content/suppl/2007/07/17/jpet.107.127365.DC1 Supplemental material to this article can be found at: at A PE T Jornals on O cber 2, 2017 jpet.asjournals.org D ow nladed from