Glycosylation Regulates Nanoscale Clustering of Na, K-ATPase as Revealed by Super-Resolution Microscopy

The Na, K-ATPase (NKA) is a critical transmembrane protein complex responsible for maintaining cellular electrochemical gradients. NKA is a heterotrimeric complex composed of alpha (α), beta (β), and gamma (γ) subunits. Glycosylation of the β subunit is necessary for its trafficking from the endoplasmic reticulum to the plasma membrane. While the functional role of the β subunit glycosylation is well-established, its impact on NKA organization at the cell surface remains unclear. Here, we investigate the role of glycosylation on membrane protein organization and dynamics by comparing the wild type (WT) β subunit to a glycosylation-deficient mutant. We used super-resolution microscopy to investigate the role of glycosylation for NKA membrane dynamics and nanoscale distribution. Fluorescence Recovery After Photobleaching (FRAP) revealed a reduced mobile fraction of glycosylation-deficient mutants. Single Molecule Localisation Microscopy (SMLM) showed that the mutants more frequently aggregate into larger clusters compared to the WT. Our experimental imaging data are supported by findings from molecular assembly simulations using GlycoSHIELD-MD that indicate a shielding effect of NKA β N-glycans. Taken together, these findings suggest that glycosylation not only has a role for trafficking but can also regulate the nanoscale organization of NKA in the plasma membrane. A higher degree of clustering of NKA could potentially influence its functional activity and interactions with other membrane components. VR 2020-05347, VR 2019-00217 (Swedish Research Council) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.