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Overview on solubilization and lipid reconstitution of Na,K-ATPase: enzyme kinetic and biophysical characterization.

ABSTRACT: Na,K-ATPase is a membrane protein which plays a vital role. It pumps Na+ and K+ ions across the cellular membranes using energy from ATP hydrolysis, and is responsible for maintaining the osmotic equilibrium and generating the membrane potential. Moreover, Na,K-ATPase has also been involved in cell signaling, interacting with partner proteins. Cardiotonic steroids bind specifically to Na,K-ATPase triggering a number of signaling pathways. Because of its importance, many efforts have been employed to study the structure and function of this protein. Difficulties associated with its removal from natural membranes and the concomitant search for appropriate replacement conditions to keep the protein in solution have presented a challenge that had to be overcome prior to carrying out biophysical and biochemical studies in vitro. In this review, we summarized all of the methods and techniques applied by our group in order to obtain information about Na,K-ATPase in respect to solubilization, reconstitution into mimetic system, influence of lipid composition, stability, oligomerization, and aggregation.

SUBMITTER: Yoneda JS

PROVIDER: S-EPMC7040103 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Overview on solubilization and lipid reconstitution of Na,K-ATPase: enzyme kinetic and biophysical characterization.

Yoneda Juliana Sakamoto JS Sebinelli Heitor Gobbi HG Itri Rosangela R Ciancaglini Pietro P

Biophysical reviews 20200118 1

Na,K-ATPase is a membrane protein which plays a vital role. It pumps Na<sup>+</sup> and K<sup>+</sup> ions across the cellular membranes using energy from ATP hydrolysis, and is responsible for maintaining the osmotic equilibrium and generating the membrane potential. Moreover, Na,K-ATPase has also been involved in cell signaling, interacting with partner proteins. Cardiotonic steroids bind specifically to Na,K-ATPase triggering a number of signaling pathways. Because of its importance, many eff ...[more]

PMID: 31955383

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Project description: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.

Dataset Information

Overview on solubilization and lipid reconstitution of Na,K-ATPase: enzyme kinetic and biophysical characterization.

Publications

Overview on solubilization and lipid reconstitution of Na,K-ATPase: enzyme kinetic and biophysical characterization.

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