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Интеллектуальная Система Тематического Исследования НАукометрических данных |
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Calcium imaging with genetically encoded sensor Case12: Facile analysis of alpha 7/alpha 9 nAChR mutantsстатья
Статья опубликована в высокорейтинговом журнале
Статья опубликована в журнале из списка Web of Science и/или Scopus- Авторы: Shelukhina Irina, Spirova Ekaterina, Kudryavtsev Denis, Ojomoko Lucy, Werner Markus, Methfessel Christoph, Hollmann Michael, Tsetlin Victor
- Журнал: PLoS ONE
- Том: 12
- Номер: 8
- Год издания: 2017
- Издательство: Public Library of Science
- Местоположение издательства: United States
- DOI: 10.1371/journal.pone.0181936
- Аннотация: Elucidation of the structural basis of pharmacological differences for highly homologous alpha 7 and alpha 9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for alpha 7 and alpha 9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of alpha 7/alpha 9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of alpha 7/alpha 9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat alpha 7 nAChRs, and for their mutants with homologous residues of alpha 9 nAChR incorporated at positions 117-119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of alpha 7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between alpha 7 and alpha 9 nAChRs.
- Добавил в систему: Кудрявцев Денис Сергеевич