FXYD proteins stabilize Na,K-ATPase : amplification of specific phosphatidylserine-protein interactions

Abstract

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary sub-units and modulate kinetic properties. This paper describes an additional property of FXYD proteins, as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (gamma subunit), and FXYD4 (CHIF) have been expressed in E.coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared to the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C12E8), with effectiveness FXYD1>FXYD2≥ FXYD4. Heating also inactivates E1↔E2 conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6M) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermal- or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically-bound phosphatidylserine is the primary cause of thermal- or detergent-mediated inactivation and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2 and FXYD4 co-expressed in Hela cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.