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Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABA A ) receptors.
Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABA A ) receptors.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2020 Aug 14; Vol. 295 (33), pp. 11495-11512. Date of Electronic Publication: 2020 Jun 15. - Publication Year :
- 2020
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Abstract
- Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABA <subscript>A</subscript> receptors (GABA <subscript>A</subscript> Rs) 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 GABA <subscript>A</subscript> R transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABA <subscript>A</subscript> Rs by photoaffinity labeling with [ <superscript>3</superscript> H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([ <superscript>3</superscript> H]21- p TFDBzox-AP), a potent GABA <subscript>A</subscript> R 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 <superscript>+</superscript> -α <superscript>-</superscript> 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 GABA <subscript>A</subscript> R antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABA <subscript>A</subscript> R 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 GABA <subscript>A</subscript> R β <superscript>+</superscript> -α <superscript>-</superscript> subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.<br />Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.<br /> (© 2020 Jayakar et al.)
- Subjects :
- Binding Sites
HEK293 Cells
Humans
Models, Molecular
Photoaffinity Labels analysis
Photoaffinity Labels metabolism
Pregnanolone analysis
Pregnanolone metabolism
Protein Multimerization
Protein Subunits chemistry
Protein Subunits metabolism
Receptors, GABA-A chemistry
Steroids chemistry
Receptors, GABA-A metabolism
Steroids metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 295
- Issue :
- 33
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 32540960
- Full Text :
- https://doi.org/10.1074/jbc.RA120.013452