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