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

Abstract 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 a softening of the membrane