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Genetic Code Expansion and Click-Chemistry Labeling to Visualize GABA-A Receptors by Super-Resolution Microscopy
oleh: 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
| Format: | Article |
|---|---|
| Diterbitkan: | Frontiers Media S.A. 2021-11-01 |
Deskripsi
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.