Phospholemman, a Single-Span Membrane Protein, Is an Accessory Protein of Na,K-ATPase in Cerebellum and Choroid Plexus

Phospholemman (FXYD1) is a homolog of the Na,K-ATPase γ subunit (FXYD2), a small accessory protein that modulates ATPase activity. Here we show that phospholemman is highly expressed in selected structures in the CNS. It is most abundant in cerebellum, where it was detected in the molecular layer, in Purkinje neurons, and in axons traversing the granule cell layer. Phospholemman was particularly enriched in choroid plexus, the organ that secretes CSF in the ventricles, where it colocalized with Na,K-ATPase in the apical membrane. It was also enriched, with Na,K-ATPase, in certain tanycytes or ependymal cells of the ventricle wall. Two different experimental approaches demonstrated that phospholemman physically associated with the Na,K-ATPase in cerebellum and choroid plexus: the proteins copurified after detergent treatment and co-immunoprecipitated from solubilized crude membranes using either anti-phospholemman or anti-Na,K-ATPase antibodies. Phospholemman antibodies precipitated all three Na,K-ATPase α subunit isoforms (α1–α3) from cerebellum, indicating that the interaction is not specific to a particular α isoform and consistent with the presence of phospholemman in both neurons and glia. Antibodies against the C-terminal domain of phospholemman reduced Na,K-ATPase activityin vitro without effect on Na+affinity. At least two other FXYD family members have been detected in the CNS, suggesting that additional complexity of sodium pump regulation will be found

Differential distribution of (Na, K)-ATPase α isoforms in the central nervous system

1. mRNA transcripts for three isoforms of the α subunit of (Na, K)-ATPase have been previously identified in the rat nervous system and designated α 1, α 2 and α 3.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44279/1/10571_2004_Article_BF00769038.pd

Regulation of the Cardiac Na+/K+ ATPase by Phospholemman

Hansraj Dhayan, Rajender Kumar, Andreas Kukol, ‘Regulation of the Cardiac Na+/K+ ATPase by Phospholemman’, in Sajal Chakraborti, Naranjan Dhalla, eds., Regulation of Membrane Na+-K+ ATPase, (Switzerland: Springer, 2016), ISBN 978-3-319-24748-9, eISBN 978-3-319-24750-2.Peer reviewe

Protein Phosphatase 2A Interacts with the Na+,K+-ATPase and Modulates Its Trafficking by Inhibition of Its Association with Arrestin

Background: The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na +,K +-ATPase. The catalytic subunit of the Na +,K +-ATPase includes several functional domains that determine its enzymatic and trafficking properties. Methodology/Principal Findings: Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A) catalytic C-subunit is a specific Na +,K +-ATPase interacting protein. PP-2A C-subunit interacted with the Na +,K +-ATPase, but not with the homologous sequences of the H +,K +-ATPase. We confirmed that the Na +,K +-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs) are important regulators of G-protein coupled receptor (GPCR) signaling, and they also regulate Na +,K +-ATPase trafficking through direct association. PP2A inhibits association between the Na +,K +-ATPase and arrestin, and diminishes the effect of arrestin on Na +,K +-ATPase trafficking. GRK phosphorylates the Na +,K +-ATPase and PP2A can at least partially reverse this phosphorylation. Conclusions/Significance: Taken together, these data demonstrate that the sodium pump belongs to a growing list of io

Surface Charges of the Membrane Crucially Affect Regulation of Na,K-ATPase by Phospholemman (FXYD1)

Abstract The human a1/His10-b1 isoform of Na,K-ATP-ase has been reconstituted as a complex with and without FXYD1 into proteoliposomes of various lipid compositions in order to study the effect of the regulatory subunit on the half-saturating Na? concentration (K1/2) of Na? ions for activation of the ion pump. It has been shown that the fraction of negatively charged lipid in the bilayer crucially affects the regulatory properties. At low concentrations of the nega-tively charged lipid DOPS (\10 %), FXYD1 increases K1/2 of Na? ions for activation of the ion pump. Phosphorylation of FXYD1 by protein kinase A at Ser68 abrogates this effect. Conversely, for proteoliposomes made with high concen-trations of DOPS ([10 %), little or no effect of FXYD1 on theK1/2 ofNa? ions is observed. Depending on ionic strength and lipid composition of the proteoliposomes, FXYD1 can alter the K1/2 of Na? ions by up to twofold. We propose possible molecular mechanisms to explain the regulatory effects of FXYD1 and the influence of charged lipid and protein phosphorylation. In particular, the positively charged C-terminal helix of FXYD1 appears to be highly mobile and may interactwith the cytoplasmicNdomain of thea-subunit, the interaction being strongly affected by phosphorylation at Ser68 and the surface charge of the membrane

Alternating Hemiplegia of Childhood-Related Neural and Behavioural Phenotypes in Na+,K+-ATPase α3 Missense Mutant Mice

Missense mutations in ATP1A3 encoding Na(+),K(+)-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC), a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na(+),K(+)-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N) that affects the same position in Na(+),K(+)-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na(+),K(+)-ATPase α3, including upon the K(+) pore and predicted K(+) binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality), directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na(+),K(+)-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC