Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ankyrin facilitates intracellular trafficking of alpha1-Na+-K+-ATPase in polarized cells.

ABSTRACT: Defects in ankyrin underlie many hereditary disorders involving the mislocalization of membrane proteins. Such phenotypes are usually attributed to ankyrin's role in stabilizing a plasma membrane scaffold, but this assumption may not be accurate. We found in Madin-Darby canine kidney cells and in other cultured cells that the 25-residue ankyrin-binding sequence of alpha(1)-Na(+)-K(+)-ATPase facilitates the entry of alpha(1),beta(1)-Na(+)-K(+)-ATPase into the secretory pathway and that replacement of the cytoplasmic domain of vesicular stomatitis virus G protein (VSV-G) with this ankyrin-binding sequence bestows ankyrin dependency on the endoplasmic reticulum (ER) to Golgi trafficking of VSV-G. Expression of the ankyrin-binding sequence of alpha(1)-Na(+)-K(+)-ATPase alone as a soluble cytosolic peptide acts in trans to selectively block ER to Golgi transport of both wild-type alpha(1)-Na(+)-K(+)-ATPase and a VSV-G fusion protein that includes the ankyrin-binding sequence, whereas the trafficking of other proteins remains unaffected. Similar phenotypes are also generated by small hairpin RNA-mediated knockdown of ankyrin R or the depletion of ankyrin in semipermeabilized cells. These data indicate that the adapter protein ankyrin acts not only at the plasma membrane but also early in the secretory pathway to facilitate the intracellular trafficking of alpha(1)-Na(+)-K(+)-ATPase and presumably other selected proteins. This novel ankyrin-dependent assembly pathway suggests a mechanism whereby hereditary disorders of ankyrin may be manifested as diseases of membrane protein ER retention or mislocalization.

SUBMITTER: Stabach PR

PROVIDER: S-EPMC2584975 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Extracellular Na(+) levels regulate formation and activity of the NaX/alpha1-Na(+)/K(+)-ATPase complex in neuronal cells.

Project description:MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as sensors of extracellular sodium concentration ([Na(+)]out). The mechanism underlying Na(+)-sensing involves Na(+)-flow through the NaX channel, directly regulated by the Na(+)/K(+)-ATPase α1-isoform which controls Na(+)-influx by modulating channel permeability. Together, these two partners form a complex involved in the regulation of intracellular sodium ([Na(+)]in). Here we aim to determine whether environmental changes in Na(+) could actively modulate the NaX/Na(+)/K(+)-ATPase complex activity. We investigated the complex activity using patch-clamp recordings from rat MnPO neurons and Neuro2a cells. When the rats were fed with a high-salt-diet, or the [Na(+)] in the culture medium was increased, the activity of the complex was up-regulated. In contrast, drop in environmental [Na(+)] decreased the activity of the complex. Interestingly under hypernatremic condition, the colocalization rate and protein level of both partners were up-regulated. Under hyponatremic condition, only NaX protein expression was increased and the level of NaX/Na(+)/K(+)-ATPase remained unaltered. This unbalance between NaX and Na(+)/K(+)-ATPase pump proportion would induce a bigger portion of Na(+)/K(+)-ATPase-control-free NaX channel. Thus, we suggest that hypernatremic environment increases NaX/Na(+)/K(+)-ATPase α1-isoform activity by increasing the number of both partners and their colocalization rate, whereas hyponatremic environment down-regulates complex activity via a decrease in the relative number of NaX channels controlled by the pump.

| S-EPMC4255601 | biostudies-literature

Regulation and identification of Na,K-ATPase alpha1 subunit phosphorylation in rat parotid acinar cells.

Project description:The stimulation of fluid and electrolyte secretion in salivary cells results in ionic changes that promote rapid increases in the activity of the Na,K-ATPase. In many cell systems, there are conflicting findings concerning the regulation of the phosphorylation of the Na,K-ATPase α subunit, which is the catalytic moiety. Initially, we investigated the phosphorylation sites on the α1 subunit in native rat parotid acinar cells using tandem mass spectrometry and identified two new phosphorylation sites (Ser(222), Ser(407)), three sites (Ser(217), Tyr(260), Ser(47)) previously found from large scale proteomic screens, and two sites (Ser(23), Ser(16)) known to be phosphorylated by PKC. Subsequently, we used phospho-specific antibodies to examine the regulation of phosphorylation on Ser(23) and Ser(16) and measured changes in ERK phosphorylation in parallel. The G-protein-coupled muscarinic receptor mimetic carbachol, the phorbol ester phorbol 12-myristate 13-acetate, the Ca(2+) ionophore ionomycin, and the serine/threonine phosphatase inhibitor calyculin A increased Ser(23) α1 phosphorylation. Inhibition of classical PKC proteins blocked carbachol-stimulated Ser(23) α1 subunit phosphorylation but not ERK phosphorylation, which was blocked by an inhibitor of novel PKC proteins. The carbachol-initiated phosphorylation of Ser(23) α1 subunit was not modified by ERK or PKA activity. The Na,K-ATPase inhibitor ouabain reduced and enhanced the carbachol-promoted phosphorylation of Ser(23) and Ser(16), respectively, the latter because ouabain itself increased Ser(16) phosphorylation; thus, both sites display conformational-dependent phosphorylation changes. Ouabain-initiated phosphorylation of Ser(16) α1 was not blocked by PKC inhibitors, unlike carbachol- or phorbol 12-myristate 13-acetate-initiated phosphorylations, suggesting that this site was also a substrate for a kinase other than PKC.

| S-EPMC2978561 | biostudies-literature

Release of intracellular calcium stores facilitates coxsackievirus entry into polarized endothelial cells.

Project description:Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.

| S-EPMC2951373 | biostudies-literature

Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells.

Project description:Stimulation of Na(+)/K(+)-ATPase activity in alveolar epithelial cells by cAMP involves its recruitment from intracellular compartments to the plasma membrane. Here, we studied the role of the actin molecular motor myosin-V in this process. We provide evidence that, in alveolar epithelial cells, cAMP promotes Na(+)/K(+)-ATPase recruitment to the plasma membrane by increasing the average speed of Na(+)/K(+)-ATPase-containing vesicles moving to the cell periphery. We found that three isoforms of myosin-V are expressed in alveolar epithelial cells; however, only myosin-Va and Vc colocalized with the Na(+)/K(+)-ATPase in intracellular membrane fractions. Overexpression of dominant-negative myosin-Va or knockdown with specific shRNA increased the average speed and distance traveled by the Na(+)/K(+)-ATPase-containing vesicles, as well as the Na(+)/K(+)-ATPase activity and protein abundance at the plasma membrane to similar levels as those observed with cAMP stimulation. These data show that myosin-Va has a role in restraining Na(+)/K(+)-ATPase-containing vesicles within intracellular pools and that this restrain is released after stimulation by cAMP allowing the recruitment of the Na(+)/K(+)-ATPase to the plasma membrane and thus increased activity.

| S-EPMC2773192 | biostudies-literature

Membrane targeting and intracellular trafficking of the human sodium-dependent multivitamin transporter in polarized epithelial cells.

Project description:The human sodium-dependent multivitamin transporter (hSMVT) mediates sodium-dependent uptake of biotin in renal and intestinal epithelia. To date, however, there is nothing known about the structure-function relationship or targeting sequences in the hSMVT polypeptide that control its polarized expression within epithelia. Here, we focused on the role of the COOH-terminal tail of hSMVT in the targeting and functionality of this transporter. A full-length hSMVT-green fluorescent protein (GFP) fusion protein was functional and expressed at the apical membrane in renal and intestinal cell lines. Microtubule disrupting agents disrupted the mobility of trafficking vesicles and impaired cell surface delivery of hSMVT, which was also prevented in cells treated with dynamitin (p50), brefeldin, or monensin. Progressive truncation of the COOH-terminal tail impaired the functionality and targeting of the transporter. First, biotin transport decreased by approximately 20-30% on deletion of up to 15 COOH-terminal amino acids of hSMVT, a decrease mimicked solely by deletion of the terminal PDZ motif (TSL). Second, deletions into the COOH-terminal tail (between residues 584-612, containing a region of predicted high surface accessibility) resulted in a further drop in hSMVT transport (to approximately 40% of wild-type). Third, apical targeting was lost on deletion of a helical-prone region between amino acids 570-584. We conclude that the COOH tail of hSMVT contains several determinants important for polarized targeting and biotin transport.

| S-EPMC2670647 | biostudies-literature

PGE2 upregulates the Na+/K+ ATPase in HepG2 cells via EP4 receptors and intracellular calcium.

Project description:The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP-Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.

| S-EPMC7808645 | biostudies-literature

Disruption of Ankyrin B and Caveolin-1 Interaction Sites Alters Na+,K+-ATPase Membrane Diffusion.

Project description:The Na+,K+-ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na+,K+-ATPase ?-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na+,K+-ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na+,K+-ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na+,K+-ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na+,K+-ATPase mutations and provide information about the interaction of Na+,K+-ATPase ?-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes.

| S-EPMC5700257 | biostudies-literature

Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP3 receptor in a cardiac T-tubule/SR microdomain.

Project description:We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms), Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor "dyads." E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B(+/-) cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.

| S-EPMC1287507 | biostudies-literature

The polarized distribution of Na+,K+-ATPase: role of the interaction between {beta} subunits.

Project description:The very existence of higher metazoans depends on the vectorial transport of substances across epithelia. A crucial element of this transport is the membrane enzyme Na(+),K(+)-ATPase. Not only is this enzyme distributed in a polarized manner in a restricted domain of the plasma membrane but also it creates the ionic gradients that drive the net movement of glucose, amino acids, and ions across the entire epithelium. In a previous work, we have shown that Na(+),K(+)-ATPase polarity depends on interactions between the beta subunits of Na(+),K(+)-ATPases located on neighboring cells and that these interactions anchor the entire enzyme at the borders of the intercellular space. In the present study, we used fluorescence resonance energy transfer and coprecipitation methods to demonstrate that these beta subunits have sufficient proximity and affinity to permit a direct interaction, without requiring any additional extracellular molecules to span the distance.

| S-EPMC2893986 | biostudies-literature

Ankyrin binds to two distinct cytoplasmic domains of Na,K-ATPase alpha subunit.

Project description:Ankyrin has emerged as a ubiquitous protein linking integral membrane transport proteins such as Na,K-ATPase to an underlying spectrin cytoskeleton. This interaction is mediated by the alpha subunit of Na,K-ATPase; however, the nature of the ankyrin binding site in Na,K-ATPase is unknown. As a step to determine the mechanism of this interaction, the ankyrin binding region of human erythrocyte spectrin and each of five putative cytoplasmic domains of the Na,K-ATPase alpha subunit have been prepared as recombinant fusion proteins in bacteria and analyzed for their interaction with erythrocyte and kidney ankyrin (Ank1 and Ank3, respectively) in vitro. Spectrin binds both Ank1 and Ank3 avidly, as expected. Two of the Na,K-ATPase domains, immobilized on a bioaffinity column, also interact specifically with both of these ankyrins. These ATPase domains are encoded by codons 140-290 (domain II) and 345-784 (domain III), with domain II displaying the greatest apparent affinity. Sequences in domain II are highly conserved between species and isoforms of Na,K-ATPase and are homologous to a cytoplasmic domain in H,K-ATPase and to a limited region of sequence in Ca-ATPase. Conversely, domain II shares no significant homology with other ankyrin binding proteins such as band 3 and Na(+)-channel proteins. These results identify a clear function for a conserved but previously not understood region of the alpha subunit of Na,K-ATPase and suggest that the interaction of ankyrin with membrane transport proteins may involve complex tertiary structural determinants not easily deduced from the primary sequence.

| S-EPMC43495 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data