Direct Visualization of Na,K-ATPase Clustering by 3D DNA-PAINT MINFLUX Nanoscopy

The sodium–potassium pump (Na,K-ATPase) is an essential plasma-membrane transporter that consumes 20%–30% of cellular energy, and up to approximately 50% in the kidneys. Whether it assembles into defined oligomers in its native membrane has been debated for decades. Resolving this question demands imaging precision at molecular length scales. Here, we combine 3D MINFLUX nanoscopy with DNA-PAINT to map EGFP-tagged Na,K-ATPase α1 and β1 subunits in mammalian cells with 3–5 nm localization precision. We introduce two point-cloud analysis tools: a data-driven algorithm that assigns localizations to the apical or basal membrane, removing trans-membrane artifacts, and a sequential nearest-neighbor analysis that estimates oligomer stoichiometry without structural priors. Both subunits show preferential nearest-neighbor distances of approximately 7 nm, direct evidence of dimerization, and are further organized into nanoclusters of approximately 7 to 12 molecules. These results provide direct structural evidence of Na,K-ATPase self-assembly and a framework for quantifying membrane-protein clustering at single-molecule resolution.