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Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors.
Jayakar, Selwyn S; Chiara, David C; Zhou, Xiaojuan; Wu, Bo; Bruzik, Karol S; Miller, Keith W; Cohen, Jonathan B.
Afiliación
  • Jayakar SS; Department of Neurobiology, Harvard Medical School, Boston, Massachusetts.
  • Chiara DC; Department of Neurobiology, Harvard Medical School, Boston, Massachusetts.
  • Zhou X; Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts.
  • Wu B; Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois.
  • Bruzik KS; Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois.
  • Miller KW; Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital, Boston, Massachusetts.
  • Cohen JB; Department of Neurobiology, Harvard Medical School, Boston, Massachusetts jonathan_cohen@hms.harvard.edu.
J Biol Chem ; 295(33): 11495-11512, 2020 08 14.
Article en En | MEDLINE | ID: mdl-32540960
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 α1ß3 and α1ß3γ2 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 ß subunit M4 (ß3Pro-415, ß3Leu-417, and ß3Thr-418) and M3 (ß3Arg-309) helices located at the base of a pocket in the ß+-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 3ß-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 ß+-α- subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Esteroides / Receptores de GABA-A Límite: Humans Idioma: En Revista: J Biol Chem Año: 2020 Tipo del documento: Article Pais de publicación: Estados Unidos
Texto completo
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XML
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Esteroides / Receptores de GABA-A Límite: Humans Idioma: En Revista: J Biol Chem Año: 2020 Tipo del documento: Article Pais de publicación: Estados Unidos