Spatial distribution and activity of Na +/K +-ATPase in lipid bilayer membranes with phase boundaries

• To reconstitute Na+,K+-ATPase (NKA) in GUVs of complex lipid mixture.
• Lipid compositional heterogeneity in GUVs containing NKA is quantified.
• NKA display membrane-phase dependent activity and spatial distribution.
• Imaging single NKA distribution in membranes of binary and ternary lipid mixtures.
• NKA prefer to localize at lo–ld domains boundaries.

We have reconstituted functional Na +/K +-ATPase (NKA) into giant unilamellar vesicles (GUVs) of well-defined binary and ternary lipid composition including cholesterol. The activity of the membrane system can be turned on and off by ATP. The hydrolytic activity of NKA is found to depend on membrane phase, and the water relaxation in the membrane on the presence of NKA. By collapsing and fixating the GUVs onto a solid support and using high-resolution atomic-force microscopy (AFM) imaging we determine the protein orientation and spatial distribution at the single-molecule level and find that NKA is preferentially located at l o/l d interfaces in two-phase GUVs and homogeneously distributed in single-phase GUVs. When turned active, the membrane is found to unbind from the support suggesting that the protein function leads to softening of the membrane.

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