title: Neutral phospholipids stimulate Na,K-ATPase activity: a specific lipid-protein interaction.
abstract: Membrane proteins interact with phospholipids either via an annular layer surrounding the transmembrane segments or by specific lipid-protein interactions. Although specifically bound phospholipids are observed in many crystal structures of membrane proteins, their roles are not well understood. Na,K-ATPase is highly dependent on acid phospholipids, especially phosphatidylserine, and previous work on purified detergent-soluble recombinant Na,K-ATPase showed that phosphatidylserine stabilizes and specifically interacts with the protein. Most recently the phosphatidylserine binding site has been located between transmembrane segments of αTM8-10 and the FXYD protein. This paper describes stimulation of Na,K-ATPase activity of the purified human α1β1 or α1β1FXYD1 complexes by neutral phospholipids, phosphatidylcholine, or phosphatidylethanolamine. In the presence of phosphatidylserine, soy phosphatidylcholine increases the Na,K-ATPase turnover rate from 5483 ± 144 to 7552 ± 105 (p < 0.0001). Analysis of α1β1FXYD1 complexes prepared with native or synthetic phospholipids shows that the stimulatory effect is structurally selective for neutral phospholipids with polyunsaturated fatty acyl chains, especially dilinoleoyl phosphatidylcholine or phosphatidylethanolamine. By contrast to phosphatidylserine, phosphatidylcholine or phosphatidylethanolamine destabilizes the Na,K-ATPase. Structural selectivity for stimulation of Na,K-ATPase activity and destabilization by neutral phospholipids distinguish these effects from the stabilizing effects of phosphatidylserine and imply that the phospholipids bind at distinct sites. A re-examination of electron densities of shark Na,K-ATPase is consistent with two bound phospholipids located between transmembrane segments αTM8-10 and TMFXYD (site A) and between TM2, -4, -6, -and 9 (site B). Comparison of the phospholipid binding pockets in E2 and E1 conformations suggests a possible mechanism of stimulation of Na,K-ATPase activity by the neutral phospholipid.
authors: Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel.
authors: Haim
authors: H
authors: Haviv
authors: Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel.
authors: Michael
authors: M
authors: Habeck
authors: Institute of Molecular and Cellular Biosciences, University of Tokyo, Bungkyo-ku, Tokyo 113-0032, Japan.
authors: Ryuta
authors: R
authors: Kanai
authors: Institute of Molecular and Cellular Biosciences, University of Tokyo, Bungkyo-ku, Tokyo 113-0032, Japan.
authors: Chikashi
authors: C
authors: Toyoshima
authors: Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel. Electronic address: Steven.Karlish@weizmann.ac.il.
authors: Steven J D
authors: SJD
authors: Karlish
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004583:Electrons; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008055:Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013025:Glycine max / Q000378:metabolism; D013552:Swine; D013696:Temperature; D013997:Time Factors
chemical_list: D008055:Lipids; D008565:Membrane Proteins; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D010750:Phosphoproteins; D011994:Recombinant Proteins; C069141:phospholemman; C483858:phosphatidylethanolamine; D000254:Sodium-Potassium-Exchanging ATPase