Stabilization of the α2 isoform of Na,K-ATPase by mutations in a phospholipid binding pocket.
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.
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Einat
E
Kapri-Pardes
Adriana
A
Katz
Haim
H
Haviv
Yasser
Y
Mahmmoud
Micha
M
Ilan
Irena
I
Khalfin-Penigel
Shmuel
S
Carmeli
Oded
O
Yarden
Steven J D
SJ
Karlish
D000818:Animals; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009154:Mutation; D010718:Phosphatidylserines / Q000037:antagonists & inhibitors / Q000378:metabolism; D010743:Phospholipids / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D011753:Pyrones / Q000494:pharmacology; D051381:Rats; D012508:Sarcolemma / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*
C008798:6-pentylpyrone; D007527:Isoenzymes; D008565:Membrane Proteins; D010718:Phosphatidylserines; D010743:Phospholipids; D010750:Phosphoproteins; D011753:Pyrones; C069141:phospholemman; D000254:Sodium-Potassium-Exchanging ATPase