(652.8) Direct Observation of Na,K-ATPase Oligomers in The Plasma Membrane of Living Cells by FRET-FCS

Poster Board Number: A146

Linnea Nordahl (KTH Royal Institute of Technology), Stefan Wennmalm (KTH Royal Institute of Technology), Jakob Jönsson (KTH Royal Institute of Technology), Simon Elsässer (Karolinska Institutet), Evgeny Akkuratov (University of Oxford), Hjalmar Brismar (KTH Royal Institute of Technology)

The sodium pump, Na,K-ATPase, is an integral plasma membrane protein, expressed in all eukaryotic cells. Na,K-ATPase transforms chemical energy from ATP into a gradient of Na+ and K+ over the plasma membrane by actively exporting three Na+ ions and importing two K+ ions for each hydrolyzed ATP. It is responsible for maintenance of the transmembrane Na+ gradient and is the major determinant of the membrane potential. It provides the driving force for all Na+-coupled transport processes, thereby controlling essential functions in the cell. Na,K-ATPase is formed by three subunits alpha/beta/FXYD, where alpha is the catalytic ion-transporting subunit, beta is a regulatory subunit and FXYD is accessory.

Self-interaction and oligomerization of the Na,K-ATPase alpha/beta heterodimer in cell membranes has been proposed and discussed for a long time but is still an open question. 

Here we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET-FCS, in order to detect oligomers of Na,K-ATPase. Compared to conventional cross-correlation FCCS, FRET-FCS is one to two orders of magnitude more sensitive when detecting oligomers. Moreover, FRET-FCS is inherently insensitive to unbalanced labeling, which is a great advantage during live cell measurements. 

We hypothesized that Na,K-ATPase can exist in a higher order oligomeric state and demonstrate the use of FRET-FCS to test this hypothesis 

We have introduced fluorescent labels by using expression of non-canonical amino acid modified beta subunits. The FRET pair Alexa488 and Alexa647 was directly conjugated to the beta subunits using selective click chemistry. Conventional FCS measurements of labeled cells revealed the absolute density of labeled and unlabeled Na,K-ATPase. With FRET-FCS we could observe FRET signals and FCS curves demonstrating the existence of oligomers. Positive controls for the FRET-FCS measurements were constructed by labeling alpha subunits with Alexa488 and beta subunits with Alexa647.

Furthermore, we performed Monte Carlo simulations of Na,K-ATPase, as monomer and as oligomer of increasing order, together with its ligands in a picket and fence diffusion model of the plasma membrane. The simulations suggest that oligomerization can have an impact on the net efficiency of the Na,K-ATPase measured as ATP turnover.

In conclusion we find that Na,K-ATPase can be found in the plasma membrane as oligomers. Further we discuss the consequences of oligomerization and propose that it can have a regulatory effect for the Na,K-ATPase net efficiency.

Funding: Vetenskapsramp;aring;det VR 2020-05347, VR 2019-00217