Direct measurements of neurosteroid binding to specific sites on GABA A receptors.

Autor: 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.; Department of Anesthesiology and Intensive Care Medicine, Kochi Medical School, Kochi, Japan., Qian M; Department of Developmental Biology (Pharmacology), Washington University School of Medicine, St. Louis, Missouri, USA., Xu Y; Department of Developmental Biology (Pharmacology), Washington University School of Medicine, St. Louis, Missouri, USA., Amtashar F; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA., Chen ZW; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.; Taylor Family Institute for Innovative Psychiatric Research, St. Louis, Missouri, USA., Kirkpatrick CC; Department of Chemistry, Saint Louis University, St. Louis, Missouri, USA., Bracamontes J; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA., Germann AL; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA., Akk G; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.; Taylor Family Institute for Innovative Psychiatric Research, St. Louis, Missouri, USA., Covey DF; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.; Department of Anesthesiology and Intensive Care Medicine, Kochi Medical School, Kochi, Japan.; Taylor Family Institute for Innovative Psychiatric Research, St. Louis, Missouri, USA.; Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri, USA., Evers AS; Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri, USA.; Department of Anesthesiology and Intensive Care Medicine, Kochi Medical School, Kochi, Japan.; Taylor Family Institute for Innovative Psychiatric Research, St. Louis, Missouri, USA.
Jazyk: angličtina
Zdroj: British journal of pharmacology [Br J Pharmacol] 2024 Nov; Vol. 181 (21), pp. 4229-4244. Date of Electronic Publication: 2024 Jul 08.
DOI: 10.1111/bph.16490
Abstrakt: Background and Purpose: Neurosteroids are allosteric modulators of GABA A 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 GABA A 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-α1GABA A R, a chimeric receptor containing transmembrane domains of the α 1 -GABA A 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 (3β-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-α1GABA A R 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.
(© 2024 The Author(s). British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)
Databáze: MEDLINE
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