title: Stimulation, inhibition, or stabilization of Na,K-ATPase caused by specific lipid interactions at distinct sites.
abstract: The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70-80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E1-E2 conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E2, but their detailed mechanisms of action are different. PE accelerates the rate of E1 → E2P but does not affect E2(2K)ATP → E13NaATP, whereas sphingomyelin inhibits the rate of E2(2K)ATP → E13NaATP, with very little effect on E1 → E2P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Michael
authors.initials: M
authors.lastname: Habeck
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Haim
authors.initials: H
authors.lastname: Haviv
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Adriana
authors.initials: A
authors.lastname: Katz
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Einat
authors.initials: E
authors.lastname: Kapri-Pardes
authors.affiliation: Max-Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Sophie
authors.initials: S
authors.lastname: Ayciriex
authors.affiliation: Max-Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
authors.forename: Andrej
authors.initials: A
authors.lastname: Shevchenko
authors.affiliation: Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.
authors.forename: Haruo
authors.initials: H
authors.lastname: Ogawa
authors.affiliation: Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.
authors.forename: Chikashi
authors.initials: C
authors.lastname: Toyoshima
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address: Steven.Karlish@weizmann.ac.il.
authors.forename: Steven J D
authors.initials: SJD
authors.lastname: Karlish
mesh_terms: D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry* / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D010718:Phosphatidylserines; D010743:Phospholipids; D010750:Phosphoproteins; C519327:stearoyl-oleoyl-phosphatidylserine; C069141:phospholemman; D002784:Cholesterol; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Cholesterol; Lipid-Protein Interaction; Membrane Protein; Membrane Transport; Na+/K+-ATPase; Phospholipid; Specific Lipid Sites