Expansion microscopy and biorthogonal labeling with non-canonical amino acids enables cluster analysis of Na, K-ATPase in the plasma membrane

Recent developments in expansion microscopy and techniques for site-specific fluorescent labeling of proteins opens new possibilities to study nano-scale protein distributions. We have studied the spatial distribution of Na, K-ATPase alpha and beta subunits in the plasma membrane of epithelial cells. For this we developed sample preparation protocols based on genetic code expansion, click chemistry and expansion microscopy. We transiently transfected HEK 293T cells with constructs containing the coding sequence for the Na, K-ATPase subunits (alpha1 and beta1) carrying a premature amber or ochre stop codon. Two bioorthogonal tRNA and tRNA synthetase pairs were also transfected for stop codon suppression and site-specific insertion of two unnatural amino acids, ProK and TCO*K. The unnatural amino acids were then labelled with small organic dyes using CuAAC and SPIEDAC click reactions.The dyes AF488 picolyl azide and Abberior STAR 635 tetrazine were used to label the two subunits on living cells. Expansion microscopy was performed using a modified MAP protocol, reaching an isotropic expansion of approximately 4.5-fold. Confocal airyscan microscopy of the expanded cells with <50nm resolution revealed nanoscale variations in Na, K-ATPase density and enabled detailed analysis of the proteins distribution. We propose that the spatial distribution of Na, K-ATPase in the plasma membrane has a regulatory function for the local ion pumping efficiency in the cell. VR 2020-05347, VR 2019-00217 (Swedish Research Council) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.