Stabilization of the ?2 isoform of Na,K-ATPase by mutations in a phospholipid binding pocket.

ABSTRACT: The ?2 isoform of Na,K-ATPase plays a crucial role in Ca(2+) handling, muscle contraction, and inotropic effects of cardiac glycosides. Thus, structural, functional, and pharmacological comparisons of ?1, ?2, and ?3 are of great interest. In Pichia pastoris membranes expressing human ?1?1, ?2?1, and ?3?1 isoforms, or using the purified isoform proteins, ?2 is most easily inactivated by heating and detergent (?2 ? ?3 > ?1). We have examined an hypothesis that instability of ?2 is caused by weak interactions with phosphatidylserine, which stabilizes the protein. Three residues, unique to ?2, in trans-membrane segments M8 (Ala-920), M9 (Leu-955), and M10 (Val-981) were replaced by equivalent residues in ?1, singly or together. Judged by the sensitivity of the purified proteins to heat, detergent, "affinity" for phosphatidylserine, and stabilization by FXYD1, the triple mutant (A920V/L955F/V981P, called ?2VFP) has stability properties close to ?1, although single mutants have only modest or insignificant effects. Functional differences between ?1 and ?2 are unaffected in ?2VFP. A compound, 6-pentyl-2-pyrone, isolated from the marine fungus Trichoderma gamsii is a novel probe of specific phospholipid-protein interactions. 6-Pentyl-2-pyrone inactivates the isoforms in the order ?2 ? ?3 > ?1, and ?2VFP and FXYD1 protect the isoforms. In native rat heart sarcolemma membranes, which contain ?1, ?2, and ?3 isoforms, a component attributable to ?2 is the least stable. The data provide clear evidence for a specific phosphatidylserine binding pocket between M8, M9, and M10 and confirm that the instability of ?2 is due to suboptimal interactions with phosphatidylserine. In physiological conditions, the instability of ?2 may be important for its cellular regulatory functions.

SUBMITTER: Kapri-Pardes E 

PROVIDER: S-EPMC3234826 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature