Your browser doesn't support javascript.
loading
Direct measurements of neurosteroid binding to specific sites on GABAA receptors.
Chintala, Satyanarayana M; Tateiwa, Hiroki; Qian, Mingxing; Xu, Yuanjian; Amtashar, Fatima; Chen, Zi-Wei; Kirkpatrick, Charles C; Bracamontes, John; Germann, Allison L; Akk, Gustav; Covey, Douglas F; Evers, Alex S.
Afiliación
  • Chintala SM; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.
  • Tateiwa H; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.
  • Qian M; Department of Anesthesiology and Intensive Care Medicine, Kochi Medical School, Kochi, Japan.
  • Xu Y; Department of Developmental Biology (Pharmacology), Washington University School of Medicine, St. Louis, Missouri, USA.
  • Amtashar F; Department of Developmental Biology (Pharmacology), Washington University School of Medicine, St. Louis, Missouri, USA.
  • Chen ZW; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.
  • Kirkpatrick CC; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.
  • Bracamontes J; Taylor Family Institute for Innovative Psychiatric Research, St. Louis, Missouri, USA.
  • Germann AL; Department of Chemistry, Saint Louis University, St. Louis, Missouri, USA.
  • Akk G; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.
  • Covey DF; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.
  • Evers AS; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.
Br J Pharmacol ; 181(21): 4229-4244, 2024 Nov.
Article en En | MEDLINE | ID: mdl-38978389
ABSTRACT
BACKGROUND AND

PURPOSE:

Neurosteroids are allosteric modulators of GABAA currents, acting through several functional binding sites although their affinity and specificity for each site are unknown. The goal of this study was to measure steady-state binding affinities of various neurosteroids for specific sites on the GABAA receptor. EXPERIMENTAL

APPROACH:

Two methods were developed to measure neurosteroid binding affinity (1) quenching of specific tryptophan residues in neurosteroid binding sites by the neurosteroid 17-methylketone group, and (2) FRET between MQ290 (an intrinsically fluorescent neurosteroid) and tryptophan residues in the binding sites. The assays were developed using ELIC-α1GABAAR, a chimeric receptor containing transmembrane domains of the α1-GABAA receptor. Tryptophan mutagenesis was used to identify specific interactions. KEY

RESULTS:

Allopregnanolone (3α-OH neurosteroid) was shown to bind at intersubunit and intrasubunit sites with equal affinity, whereas epi-allopregnanolone (3ß-OH neurosteroid) binds at the intrasubunit site. MQ290 formed a strong FRET pair with W246, acting as a site-specific probe for the intersubunit site. The affinity and site-specificity of several neurosteroid agonists and inverse agonists was measured using the MQ290 binding assay. The FRET assay distinguishes between competitive and allosteric inhibition of MQ290 binding and demonstrated an allosteric interaction between the two neurosteroid binding sites. CONCLUSIONS AND IMPLICATIONS The affinity and specificity of neurosteroid binding to two sites in the ELIC-α1GABAAR were directly measured and an allosteric interaction between the sites was revealed. Adaptation of the MQ290 FRET assay to a plate-reader format will enable screening for high affinity agonists and antagonists for neurosteroid binding sites.
Asunto(s)
Palabras clave
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Receptores de GABA-A / Neuroesteroides Límite: Animals / Humans Idioma: En Revista: Br J Pharmacol Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Receptores de GABA-A / Neuroesteroides Límite: Animals / Humans Idioma: En Revista: Br J Pharmacol Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Reino Unido