Abstract
The α2 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 α1, α2, and α3 are of great interest. In Pichia pastoris membranes expressing human α1β1, α2β1, and α3β1 isoforms, or using the purified isoform proteins, α2 is most easily inactivated by heating and detergent (α2 ≫ α3 > α1). We have examined an hypothesis that instability of α2 is caused by weak interactions with phosphatidylserine, which stabilizes the protein. Three residues, unique to α2, in trans-membrane segments M8 (Ala-920), M9 (Leu-955), and M10 (Val-981) were replaced by equivalent residues in α1, 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 α2VFP) has stability properties close to α1, although single mutants have only modest or insignificant effects. Functional differences between α1 and α2 are unaffected in α2VFP. 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 α2 ≫ α3 > α1, and α2VFP and FXYD1 protect the isoforms. In native rat heart sarcolemma membranes, which contain α1, α2, and α3 isoforms, a component attributable to α2 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 α2 is due to suboptimal interactions with phosphatidylserine. In physiological conditions, the instability of α2 may be important for its cellular regulatory functions.
Highlights
The ␣2 isoform of Na,K-ATPase is unstable compared with ␣1 and ␣3
Instability of ␣2—As described in the Introduction, when human ␣1 and ␣2 isoforms were expressed in P. pastoris and purified, it was found that ␣2 is much less stable than ␣1, an effect attributed to suboptimal interaction with SOPS [21]
This was done in measurements of both ouabain binding to the intact yeast membranes (Fig. 1A) and Na,KATPase activity of the purified isoform proteins (Fig. 1B)
Summary
The ␣2 isoform of Na,K-ATPase is unstable compared with ␣1 and ␣3. Results: Mutations in TM8 –10 strongly stabilize ␣2. In Pichia pastoris membranes expressing human ␣11, ␣21, and ␣31 isoforms, or using the purified isoform proteins, ␣2 is most 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. 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, single mutants have only modest or insignificant effects. A compound, 6-pentyl2-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. The instability of ␣2 may be important for its cellular regulatory functions
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