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

Alexander Kuhlemann,Andrea Barberis,Carmen Villmann,Markus Sauer,Christian Werner,Danush Taban,Sören Doose,Martina Bruno,Enrica Maria Petrini,Dieter Janzen,Dominic A Helmerich,Gerti Beliu

doi:10.3389/fnsyn.2021.727406

Alexander Kuhlemann, Andrea Barberis + Show 10 more

Open Access

https://doi.org/10.3389/fnsyn.2021.727406

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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.

Highlights

In the central nervous system, phasic and tonic inhibition is dominantly controlled by γ-aminobutyric acid type A (GABA-A) receptors
Each Amber mutant construct of GABA-A receptor α2 subunit was co-transfected with β1 and γ2 subunits in HEK-293-T cells to ensure proper surface delivery of α2 subunits, along with an orthogonal tRNA/tRNA-synthetase pair (Nikic et al, 2016)
To visualize the distribution of GABA-A receptors in the plasma membrane of HEK-293-T cells, we used single-molecule sensitive super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM) of immunolabeled and tetrazine-dye labeled receptors (S181TAG) in Total internal reflection (TIRF) mode (Haselmann et al, 2018; Siddig et al, 2020). dSTORM images revealed a homogeneous distribution of GABA-A receptors on the basal plasma membrane independent of the used labeling method

Summary

Introduction

In the central nervous system, phasic and tonic inhibition is dominantly controlled by γ-aminobutyric acid type A (GABA-A) receptors. We set out to introduce ncAA in extracellular domains of GABA-A receptor subunits by GCE in HEK-293-T cells and primary cultured neurons followed by site-specific labeling with tetrazine dyes and visualization by direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM) (Gustafsson, 2000; Heilemann et al, 2008; van de Linde et al, 2011).

Results

Conclusion