FXYD Proteins Stabilize Na,K-ATPase

Abstract

FXYDproteins are a family ofseven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K- ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property ofFXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (␥ subunit), and FXYD4 (CHIF) have been expressed in Esche- richia coli and purified. These FXYD proteins associate sponta- neously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (␣1␤1) to form ␣1␤1FXYD complexes. Compared with the control (␣1␤1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C12E8), with effectivenessFXYD1> FXYD2 > FXYD4. Heating also inactivates E1 7 E2 conforma- tional changes and cation occlusion, and FXYD1 protects strongly. Incubation of ␣1␤1or ␣1␤1FXYD complexes with guanidinium chloride (up to 6 M) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus,