Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy

Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type...

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Autores principales:	Alexander Kuhlemann, Gerti Beliu, Dieter Janzen, Enrica Maria Petrini, Danush Taban, Dominic A. Helmerich, Sören Doose, Martina Bruno, Andrea Barberis, Carmen Villmann, Markus Sauer, Christian Werner
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	super-resolution microscopy (SRM) click-chemistry dSTORM GABA-A receptor genetic code expansion Neurosciences. Biological psychiatry. Neuropsychiatry RC321-571
Acceso en línea:	https://doaj.org/article/e70170de723941fa9ef714c626af7fbd
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spelling	oai:doaj.org-article:e70170de723941fa9ef714c626af7fbd2021-12-01T08:21:28ZGenetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy1663-356310.3389/fnsyn.2021.727406https://doaj.org/article/e70170de723941fa9ef714c626af7fbd2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fnsyn.2021.727406/fullhttps://doaj.org/toc/1663-3563Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.Alexander KuhlemannGerti BeliuGerti BeliuDieter JanzenEnrica Maria PetriniDanush TabanDominic A. HelmerichSören DooseMartina BrunoAndrea BarberisCarmen VillmannMarkus SauerChristian WernerFrontiers Media S.A.articlesuper-resolution microscopy (SRM)click-chemistrydSTORMGABA-A receptorgenetic code expansionNeurosciences. Biological psychiatry. NeuropsychiatryRC321-571ENFrontiers in Synaptic Neuroscience, Vol 13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	super-resolution microscopy (SRM) click-chemistry dSTORM GABA-A receptor genetic code expansion Neurosciences. Biological psychiatry. Neuropsychiatry RC321-571
spellingShingle	super-resolution microscopy (SRM) click-chemistry dSTORM GABA-A receptor genetic code expansion Neurosciences. Biological psychiatry. Neuropsychiatry RC321-571 Alexander Kuhlemann Gerti Beliu Gerti Beliu Dieter Janzen Enrica Maria Petrini Danush Taban Dominic A. Helmerich Sören Doose Martina Bruno Andrea Barberis Carmen Villmann Markus Sauer Christian Werner Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
description	Fluorescence labeling of difficult to access protein sites, e.g., in confined compartments, requires small fluorescent labels that can be covalently tethered at well-defined positions with high efficiency. Here, we report site-specific labeling of the extracellular domain of γ-aminobutyric acid type A (GABA-A) receptor subunits by genetic code expansion (GCE) with unnatural amino acids (ncAA) combined with bioorthogonal click-chemistry labeling with tetrazine dyes in HEK-293-T cells and primary cultured neurons. After optimization of GABA-A receptor expression and labeling efficiency, most effective variants were selected for super-resolution microscopy and functionality testing by whole-cell patch clamp. Our results show that GCE with ncAA and bioorthogonal click labeling with small tetrazine dyes represents a versatile method for highly efficient site-specific fluorescence labeling of proteins in a crowded environment, e.g., extracellular protein domains in confined compartments such as the synaptic cleft.
format	article
author	Alexander Kuhlemann Gerti Beliu Gerti Beliu Dieter Janzen Enrica Maria Petrini Danush Taban Dominic A. Helmerich Sören Doose Martina Bruno Andrea Barberis Carmen Villmann Markus Sauer Christian Werner
author_facet	Alexander Kuhlemann Gerti Beliu Gerti Beliu Dieter Janzen Enrica Maria Petrini Danush Taban Dominic A. Helmerich Sören Doose Martina Bruno Andrea Barberis Carmen Villmann Markus Sauer Christian Werner
author_sort	Alexander Kuhlemann
title	Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
title_short	Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
title_full	Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
title_fullStr	Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
title_full_unstemmed	Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
title_sort	genetic code expansion and click-chemistry labeling to visualize gaba-a receptors by super-resolution microscopy
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/e70170de723941fa9ef714c626af7fbd
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Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy

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