Specific phospholipid binding to Na,K-ATPase at two distinct sites

Significance Activity and structural integrity of membrane proteins can be regulated by physical properties of the bilayer or specific lipid–protein interactions. This work shows that key properties of the Na,K-ATPase are modulated independently by specific binding of 18:0/18:1 phosphatidylserine (PS) and 18:0/20:4 phosphatidylcholine (PC) in the absence of a bilayer. PS stabilizes the protein, and PC/ phosphatidylethanolamine (PE) stimulates Na,K-ATPase activity. We characterized effects of both types of phospholipids by kinetic approaches, mutant analyses, and native MS. Modulation of Na,K-ATPase function by PS and PC/PE is defined by the phospholipid structural specificity, binding stoichiometry within two specific binding sites, and the kinetic mechanism. We provide detailed mechanistic insights, potentially with important implications for physiological regulation of active Na and K transport.