Click chemistry, FRET-FCS, and expansion microscopy reveal that Na, K-ATPase forms complexes in the plasma membrane

Abstract

In order to study the spatial distribution of Na, K-ATPase alpha and beta subunits in the plasma membrane of epithelial cells we have developed sample preparation protocols based on genetic code expansion, click chemistry and expansion microscopy. HEK 293T cells were transiently transfected with constructs containing the coding sequence for 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. The unnatural amino acids were then labelled with small organic dyes using CuAAC and SPIEDAC click reactions. Care was taken to keep the transfection and expression levels low.In living cells, we used Fluorescence Correlation Spectroscopy (FCS) and FRET to assess the density of click-labeled and endogenous [SE1] Na, K-ATPase in the plasma membrane. The dyes Alexa488 and Alexa647 were used as a FRET sensor for homo- and heterodimer analysis and, using FRET-FCS, we could detect the oligomerization of alpha and beta in a complex containing two alpha and two beta subunits in a heterodimer.FCS measurements in cells where both the alpha and beta subunits were labeled was used to calculate the absolute density of endogenous and exogenous Na, K-ATPase in the plasma membrane. To detect oligomerization, we used cells where the beta subunit was labeled with a 50/50 mix of Alexa488 and Alexa647. We successfully detected FRET signals and an analysis of the FRET-FCS data revealed a high density of oligomers. FRET-FCS is, compared to conventional cross-correlation FCCS, one to two orders of magnitude more sensitive for the detection of oligomers. FRET-FCS is also inherently insensitive to heterogenous labeling efficiencies, which is a great advantage during live cell measurementsExpansion 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 local variations in Na, K-ATPase density and enabled detailed cluster analysis.In conclusion we demonstrate that the density of Na, K-ATPase has large local variations and that the Na, K-ATPase oligomerizes in the plasma membrane. We propose that this oligomerization can have a regulatory function for the net efficiency of Na, K-ATPase 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.