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.
Highlights
Two sites for phospholipids; Biochemical and Structural Data— We propose here that there are two specific phospholipid binding sites on the ␣ subunit of Na,K-ATPase, one primarily for acid phospholipids, SOPS, that stabilizes the protein, and the other for neutral phospholipids PC or PE that mediates increased Na,K-ATPase activity
The biochemical evidence for the SOPS site within the crevice located between TM8 –10 of the ␣ subunit and the transmembrane segment of the FXYD protein has been presented and extensively discussed in previous publications (29 –33)
Compared with SOPS alone, the neutral phospholipids do not stabilize but somewhat destabilize the protein to thermal inactivation. These features, and the structural selectivity for this effect, DLPC ϭ DLPE Ͼ SLPC Ͼ SOPC, suggest strongly that there is a separate site for the neutral phospholipids
Summary
Haim Haviv‡1, Michael Habeck‡, Ryuta Kanai§, Chikashi Toyoshima§ and Steven J. D. Karlish‡2 From the ‡Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel and §Institute of Molecular and Cellular Biosciences, University of Tokyo, Bungkyo-ku, Tokyo 113-0032, Japan
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
