Activity and structural integrity of membrane proteins can be regulated either by physical properties of the bilayer or by site-­‐specific interactions. For Na,K-­‐ATPase, lipids bound to the transmembrane domain were observed in crystal structures yet effects of specific lipid binding are poorly understood.
We addressed this question by combining site directed mutagenesis, biochemical analyses and native mass spectrometry and studied the effect of lipids on stability and activity of recombinant Na,K-­‐ATPase. Phosphatidylserine (PS) and cholesterol stabilize the  Na-­‐pump  against  thermal and  detergent  mediated  inactivation  but  do  not  per  se affect activity. ATPase activity is
stimulated by polyunsaturated phosphatidylcholine   or –ethanolamine    (PC/PE),    which    results    from    the acceleration    of    the E1P-­‐E2P conformational transition. Both effects occur in detergent in the absence of a bilayer.
Furthermore, they are independent of each other and can be selectively abrogated by mutation of  lysine residues at the cytoplasm-­‐membrane interface. Thus, binding sites for PS and PC/PE were identified. These findings support the idea of functional specific phospholipid-­‐Na,K-­‐ATPase 
interactions.  We  show  specific  lipid  binding  by  native  MS, which  revealed  that  one molecule  each  of  PS  and  PC  can  bind  specifically  to  the  Na-­‐ pump. Functional properties of detergent soluble Na,K-­‐ATPase with PS, cholesterol and PC are remarkably similar to properties of renal enzyme highlighting the importance   ofspecific interactions over bulk bilayer interactions.