Abstract
Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABAAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M4 (β3Pro-415, β3Leu-417, and β3Thr-418) and M3 (β3Arg-309) helices located at the base of a pocket in the β+-α- subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABAAR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABAAR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABAAR β+-α- subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.
Highlights
Endogenous neurosteroids, including allopregnanolone (3a5a-P) and pregnanolone (3a5b-P), can produce anxiolytic, sedative, and anti-convulsive effects [1, 2], and their synthetic analogs are in development as general anesthetics and for treatment of epilepsy, anxiety, depression, and other mood disorders [3, 4]
By use of competition photolabeling with a panel of steroid g-aminobutyric acid type A receptors (GABAAR) positive allosteric modulator (PAM) and inhibitors, we provide a first definition of the structural determinants important for high affinity binding to this site
In equilibrium binding assays with the agonist [3H]muscimol, GABAAR PAMs, including steroids and other general anesthetics, enhance binding by increasing the fraction of GABAARs in a desensitized state that binds [3H]muscimol with high affinity [28]. 21-pTFDBzox-AP was shown previously to enhance [3H] muscimol binding to expressed a1b3 and a1b3g2 GABAARs in membranes, and after purification in detergent/lipid micelles, with concentrations producing half-maximal enhancement (EC50, 0.2-0.5 mM) similar to those for 3a5a-P, 3a5b-P, and alphaxalone [27]. We extended these studies by characterizing [3H]muscimol binding to a1b3 GABAARs in the presence of steroids that act as GABAAR negative allosteric modulators, inhibiting GABA responses noncompetitively: the 3b-epimers of 3a5a-P, 3a5b-P, and alphaxalone, and two 3b-sulfated steroids (PS and dehydroepiandrosterone sulfate (DHEAS)) [10, 12, 29, 30] (Fig. 2 and Table 1)
Summary
Endogenous neurosteroids, including allopregnanolone (3a5a-P) and pregnanolone (3a5b-P), can produce anxiolytic, sedative, and anti-convulsive effects [1, 2], and their synthetic analogs are in development as general anesthetics and for treatment of epilepsy, anxiety, depression, and other mood disorders [3, 4] These neuroactive steroids act at submicromolar concentrations as potent positive allosteric modulators (PAMs) of edu. Steroids with a 3a-OH, including 3a5a-P and the anesthetic alphaxalone, act as PAMs, whereas their 3b-OH epimers (3b5a-P and betaxalone) at higher concentrations inhibit GABA responses [9,10,11,12] This structural specificity provided early evidence that steroids might interact with specific binding sites in GABAARs, identification and characterization of which would prove important for the development of novel steroid-based therapeutic agents. By use of competition photolabeling with a panel of steroid GABAAR PAMs and inhibitors, we provide a first definition of the structural determinants important for high affinity binding to this site
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
