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Functional interactions of phospholemman (PLM) (FXYD1) with Na+,K+-ATPase. Purification of alpha1/beta1/PLM complexes
Yael Lifshitz1, Moshit Lindzen, Haim Garty
1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Insights
Human FXYD1 (phospholemman, PLM) modulates Na+,K+-ATPase activity by altering ion affinity, with effects dependent on its phosphorylation state. This protein plays a key role in regulating ion transport in muscle cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Physiology
Background:
- The Na+,K+-ATPase is a crucial ion pump in cell membranes, responsible for maintaining electrochemical gradients.
- FXYD1, also known as phospholemman (PLM), is a small membrane protein that interacts with the Na+,K+-ATPase.
- Understanding the regulatory role of FXYD1 on Na+,K+-ATPase function is important for comprehending cellular ion homeostasis, particularly in muscle tissues.
Purpose of the Study:
- To investigate the functional impact of human FXYD1 (PLM) on the Na+,K+-ATPase.
- To determine how PLM affects the kinetic properties of the Na+,K+-ATPase, including ion affinity and turnover rate.
- To explore the influence of PLM phosphorylation on its interaction with and regulation of the Na+,K+-ATPase.
Main Methods:
- Expression of human FXYD1 (PLM) and porcine Na+,K+-ATPase alpha1/His10-beta1 subunits in Pichia pastoris.
- Purification of soluble alpha1/beta1/PLM complexes using metal chelate chromatography.
- Functional characterization of purified Na+,K+-ATPase complexes using enzyme kinetics assays.
- Expression of rat alpha1 and PLM in HeLa cells to compare functional effects in a mammalian system.
- Analysis of PLM phosphorylation status using specific antibodies.
Main Results:
- PLM moderately decreased the apparent affinity for Na+ ions (K0.5) but did not affect the turnover rate or Km for ATP in P. pastoris.
- PLM significantly reduced the K0.5 for Na+ ions in P. pastoris, indicating enhanced activation by Na+.
- In HeLa cells, rat PLM increased the K0.5 for Na+ ions, while a chimeric PLM molecule reduced it, suggesting context-dependent effects.
- Opposite functional effects in P. pastoris and HeLa cells correlated with the phosphorylation state of PLM (Ser68).
Conclusions:
- FXYD1 (PLM) interacts with Na+,K+-ATPase alpha1/beta1 subunits and modulates its activity.
- The functional effects of PLM on Na+,K+-ATPase are dependent on its phosphorylation status, particularly at Ser68.
- PLM's role in regulating Na+,K+-ATPase activity is complex and may involve interactions at multiple sites, with implications for cardiac and skeletal muscle function.
Abstract:
Human FXYD1 (phospholemman, PLM) has been expressed in Pichia pastoris with porcine alpha1/His10-beta1 subunits of Na+,K+-ATPase or alone. Dodecyl-beta-maltoside-soluble complexes of alpha1/beta1/PLM have been purified by metal chelate chromatography, either from membranes co-expressing alpha1,His10-beta1, and PLM or by in vitro reconstitution of PLM with alpha1/His10-beta1 subunits. Comparison of functional properties of purified alpha1/His10-beta1 and alpha1/His10-beta1/PLM complexes show that PLM lowered K0.5 for Na+ ions moderately (approximately 30%) but did not affect the turnover rate or Km of ATP for activating Na+,K+-ATPase activity. PLM also stabilized the alpha1/His10-beta1 complex. In addition, PLM markedly (>3-fold) reduced the K0.5 of Na+ ions for activating Na+-ATPase activity. In membranes co-expressing alpha1/His10-beta1 with PLM the K0.5 of Na+ ions was also reduced, compared with the control, excluding the possibility that detergent or lipid in purified complexes compromise functional interactions. When expressed in HeLa cells with rat alpha1, rat PLM significantly raised the K0.5 of Na+ ions, whereas for a chimeric molecule consisting of transmembranes segments of PLM and extramembrane segments of FXYD4, the K0.5 of Na+ ions was significantly reduced, compared with the control. The opposite functional effects in P. pastoris and HeLa cells are correlated with endogenous phosphorylation of PLM at Ser68 or unphosphorylated PLM, respectively, as detected with antibodies, which recognize PLM phosphorylated at Ser68 (protein kinase A site) or unphosphorylated PLM. We hypothesize that PLM interacts with alpha1/His10-beta1 subunits at multiple locations, the different functional effects depending on the degree of phosphorylation at Ser68. We discuss the role of PLM in regulation of Na+,K+-ATPase in cardiac or skeletal muscle cells.
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