Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
PLoS One
2012 Jan 01;74:e34879. doi: 10.1371/journal.pone.0034879.
Show Gene links
Show Anatomy links
Functional interaction between CFTR and the sodium-phosphate co-transport type 2a in Xenopus laevis oocytes.
Bakouh N
,
Chérif-Zahar B
,
Hulin P
,
Prié D
,
Friedlander G
,
Edelman A
,
Planelles G
.
???displayArticle.abstract???
BACKGROUND: A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF) when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a) functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.
METHODOLOGY/FINDINGS: NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi) was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC) had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.
CONCLUSION/PERSPECTIVES: We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis.
???displayArticle.pubmedLink???
22514683
???displayArticle.pmcLink???PMC3325942 ???displayArticle.link???PLoS One
Figure 1. Phosphate-induced current in X. laevis oocytes.Phosphate-induced current (1 mM of Pi added in ND96) was measured at holding potential (Vc) of −50 mV in oocytes expressing NPT2a or CFTR alone or co-expressing NPT2a and CFTR; oocytes injected with water were used as control oocytes. A: Original tracings showing the current induced by the addition of 1 mM Pi (indicated by black bars) in the superfusing medium. The type of oocytes is indicated above the tracings. B: Summary of the results (means ± SEM) of the effect of 1 mM Pi in the different types of oocytes (indicated below the columns). Significance of the results *: P<0.05
Figure 2. Kinetic analysis of inorganic phosphate transport in oocytes expressing NPT2a or co-expressing NPT2a and CFTR.Phosphate-induced currents (IPi) were measured (Vc = −50 mV) in NPT2a-oocytes (empty circles) and in NPT2a+CFTR-oocytes (filled circles). The dashed lines through the points (means ± SEM from n = 6 to 14, N = 5) were calculated using SigmaPlot software (Systat software Inc., San Jose, CA). A: IPi was induced by increasing Pi concentration in ND 96, pH 7.5, as indicated in abscissa and normalized against extrapolated maximal current (IPimax). Data were fitted to Michaelis-Menten equation. Results, as means ± SD, were as follows: the apparent concentration of Pi substrate (Km) giving the half IPimax was not changed by CFTR expression (Km = 0.07± 0.02 vs 0.06 ± 0.02 mM in NPT2a- and NPT2a+CFTR-oocytes, respectively), but IPimax was decreased by 40±3% (P <0.05). B: IPi was induced by 1 mM Pi at increasing Na+ concentrations (equimolary substituted by choline+, pH 7.5) and normalized against IPimax. Data were fitted to the modified Hill equation. Results, as means ± SD, were as follows: the apparent Km for Na+ substrate was 40.7 ± 14.6 in NPT2a-oocytes, not different from 41.8 ± 13.4 mM in NPT2a+CFTR-oocytes; IPimax was decreased by 39±7% in NPT2a+CFTR-oocytes compared to NPT2a-oocytes (P < 0.05). Inset: Effect of varying extracellular pH from 7.0 to 8.0 on the current induced by 1 mM Pi in NPT2a-oocytes (white column) and in NPT2a+CFTR-oocytes (black column). IPi was normalized against the Pi-induced current measured at pH 7.5 in NPT2a-oocytes from the same batch of oocytes. Results are shown as means± SEM, n = 5 oocytes of each type.
Figure 3. Immunodetection of Myc-NPT2a protein expressed in Myc-NPT2a- and Myc-NPT2a+CFTR-oocytes.A: Myc-NPT2a expression in Myc-NPT2a- and Myc-NPT2a+CFTR-oocytes (total or biotinylated proteins) was analyzed by Western blot using a polyclonal Myc antibody. H2O-injected oocytes were used as control. Actin expression was used as loading control of total proteins. The molecular weight of Myc-NPT2a is indicated. B: Left part: Quantification of the staining intensity of Myc-NPT2a in total lysate over the staining intensity of actin from Myc-NPT2a-oocytes (white column) and NPT2a+CFTR-oocytes (black column). Results are expressed as means ± SEM (N = 4). Right part: Quantification of the staining intensity of Myc-NPT2a cell surface expression from Myc-NPT2a+CFTR oocytes (black column) normalized against the staining intensity of Myc-NPT2a cell surface expression from Myc-NPT2a-oocytes. Results are expressed as means ± SEM (N = 4); *: P<0.05.
Figure 4. Effect of a permeant cAMP analog on the phosphate-induced current in NPT2a-oocytes or NPT2a+CFTR-oocytes.Phosphate-induced current (IPi) was measured in voltage-clamped (−50 mV) NPT2a- or co-and NPT2a+CFTR-oocytes. A. Original tracings obtained from a NPT2a-oocyte (left), and from a NPT2a+CFTR-oocyte (right), exposed in a reversible manner to 1 mM Pi, as indicated by a black bar. As indicated below the tracings, IPi was first induced in basal condition, basalIPi (superfusion of ND96 supplemented with 1 mM Pi), then in an experimental condition, exptlIPi, herein after 10 min exposure to 8 Bromoadenosine-3′5′-cyclic monophosphate (8-Br-cAMP, 100 µM). The break in each trace represents a ∼ 9 min exposure to the cAMP agonist that is not shown. B. Summary of the results (means ± SEM) of the effect of 8-Br-cAMP, 100 µM, on IPi in NPT2a-oocytes (n = 8, white columns) and in NPT2a+CFTR-oocytes (n = 8, black columns) from N = 3 experiments. Measured IPi were normalized against basalIPi from NPT2a-oocytes from the same batch of oocytes. The significance of the difference between Pi-induced currents measured in basal or in experimental conditions in NPT2a+CFTR-oocytes and basalIPi from NPT2a-oocytes was analyzed using unpaired Student’s t-test (*: P < 0.05). The significance of the difference between basalIPi and exptlIPi within the same type of oocytes was assessed using paired Student’s t-test (#: P < 0.05).
Figure 5. Effects of agonists of EPAC and PKA pathways on NPT2a function and surface expression.Phosphate-induced currents (IPi) were evoked in voltage-clamped (–50 mV) cells by the exposure of NPT2a-oocytes (white columns), or NPT2a+CFTR-oocytes (black columns) to 1 mM Pi. As above, IPi was first induced in basal condition, basalIPi, then after the activation of a cAMP-dependent signaling pathway, exptlIPi, during 10 min (panels A and B). Results are as means ± SEM. IPi were normalized against basalIPi from NPT2a-oocytes from the same batch of oocytes. The significance of the difference between basalIPi from NPT2a-oocytes and basalIPi or exptlIPi from NPT2a+CFTR-oocytes was analyzed using unpaired Student’s t-test (*: P < 0.05). The significance of the difference between basalIPi and exptlIPi within the same type of oocytes was assessed using paired Student’s t-test (#: P < 0.05). A: Effect of para-Chlorophenylthio-2′-O-methyladenosine-3′, 5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP, 25 µM), an activator on the EPAC pathway on IPi in NPT2a- and NPT2a+CFTR-oocytes (n = 9 for each type; N = 2). B: Effect of N6-monobutyryladenosine-3′, 5′-cyclic monophosphate (6-MB-cAMP, 25 µM), an activator of the PKA pathway on IPi in NPT2a- and NPT2a+CFTR-oocytes (n = 19 and n = 22, respectively; N = 6).
Figure 6. Effect of PKA stimulation on NPT2a and CFTR cell surface expression.Myc-NPT2a- and Myc-NPT2a+CFTR-oocytes from a same batch were kept in control condition or submitted to 25 µM of 6-MB-cAMP for 15 min, as indicated below the panel. Control oocytes were H2O-injected. A: Effect of PKA stimulation on NPT2a cell surface expression. Left: Cell surface biotinylated proteins were probed with an anti-Myc antibody; the molecular weight of Myc-NPT2a is indicated on the figure. Right: Results from 4 separate experiments, were quantified. The staining intensity of Myc-NPT2a cell surface expression from Myc-NPT2a+CFTR oocytes (black columns) was normalized against the staining intensity of Myc-NPT2a cell surface expression from Myc-NPT2a-oocytes (white columns) in basal condition. The significance of the difference between the results was assessed using unpaired (*: P < 0.05) or paired (#: P < 0.05) Student’s t-test. B: Effect of PKA stimulation on CFTR cell surface expression. Cell surface biotinylated proteins were probed with an anti-CFTR antibody; the molecular weight of CFTR is indicated on the figure. Similar results were obtained in 2 independent experiments.
Figure 7. NPT2a and CFTR expressed in X. laevis oocytes co-immunoprecipitate.Representative experiment showing the co-immunoprecipitation of NPT2a and CFTR after their expression in X. laevis oocytes. Proteins from oocytes co-expressing Myc-NPT2a and CFTR were immunoprecipitated with MM13-4 Ab, or with IgG1 Ab used as a negative control. They were probed with the anti-Myc antibody. The molecular weight of the detected protein is indicated Similar results were obtained in N = 3 independent experiments.
Figure 8. Effect of injecting NEG2 peptide on NPT2a function.The current induced by 1 mM phosphate (IPi) was measured in voltage-clamped (−50 mV) NPT2a-oocytes and was measured in oocytes injected either with the NEG2 peptide (black column, n = 12), with a scrambled peptide (sNEG2, grey column, n = 9), or in non-injected oocytes (white column, n = 10). IPi was measured in basal condition (basalIPi, measured in ND96 medium) then under an experimental stimulating condition (exptlIPi, measured after a 10 min-incubation in ND96 supplemented with forskolin, 1µM, and isobutylmethylxanthine, 100 µM, Forsk+IBMX). Results are presented as means ± SEM from N = 4 experiments. IPi were normalized against basalIPi from control (non injected with a peptide) NPT2a-oocytes from the same batch of oocytes. The significance of the difference was analyzed using unpaired Student’s t-test (*: P < 0.05) or paired Student’s t-test (#: P < 0.05).
Ahmad,
Role of vacuolar ATPase in the trafficking of renal type IIa sodium-phosphate cotransporter.
2011, Pubmed
Ahmad,
Role of vacuolar ATPase in the trafficking of renal type IIa sodium-phosphate cotransporter.
2011,
Pubmed
Ali,
The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels.
1998,
Pubmed
,
Xenbase
Bachhuber,
Cl- interference with the epithelial Na+ channel ENaC.
2005,
Pubmed
,
Xenbase
Bankir,
Extracellular cAMP inhibits proximal reabsorption: are plasma membrane cAMP receptors involved?
2002,
Pubmed
Bertrand,
The role of regulated CFTR trafficking in epithelial secretion.
2003,
Pubmed
Bronckers,
The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in maturation stage ameloblasts, odontoblasts and bone cells.
2010,
Pubmed
Chappe,
Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA.
2003,
Pubmed
Chernova,
Acute regulation of the SLC26A3 congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes.
2003,
Pubmed
,
Xenbase
Cunningham,
Signaling pathways utilized by PTH and dopamine to inhibit phosphate transport in mouse renal proximal tubule cells.
2009,
Pubmed
Devuyst,
Chloride channels in the kidney: lessons learned from knockout animals.
2002,
Pubmed
Déliot,
Parathyroid hormone treatment induces dissociation of type IIa Na+-P(i) cotransporter-Na+/H+ exchanger regulatory factor-1 complexes.
2005,
Pubmed
Forster,
Protein kinase C activators induce membrane retrieval of type II Na+-phosphate cotransporters expressed in Xenopus oocytes.
1999,
Pubmed
,
Xenbase
Föller,
PKB/SGK-resistant GSK3 enhances phosphaturia and calciuria.
2011,
Pubmed
,
Xenbase
Gibney,
The association of nephrolithiasis with cystic fibrosis.
2003,
Pubmed
Harris,
A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.
2007,
Pubmed
,
Xenbase
Hayes,
Protein kinase C consensus sites and the regulation of renal Na/Pi-cotransport (NaPi-2) expressed in XENOPUS laevis oocytes.
1995,
Pubmed
,
Xenbase
Holz,
Cell physiology of cAMP sensor Epac.
2006,
Pubmed
Honegger,
Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC).
2006,
Pubmed
Hoppe,
Beta-receptors in resistance to phosphaturic effect of PTH in respiratory alkalosis.
1988,
Pubmed
Howard,
cAMP-regulated trafficking of epitope-tagged CFTR.
1996,
Pubmed
,
Xenbase
Jankowski,
The opossum kidney cell type IIa Na/P(i) cotransporter is a phosphoprotein.
2001,
Pubmed
Ji,
Up-regulation of acid-gated Na(+) channels (ASICs) by cystic fibrosis transmembrane conductance regulator co-expression in Xenopus oocytes.
2002,
Pubmed
,
Xenbase
Ji,
The cytosolic termini of the beta- and gamma-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel.
2000,
Pubmed
,
Xenbase
Jouret,
Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney.
2007,
Pubmed
Katz,
Microscopic nephrocalcinosis in cystic fibrosis.
1988,
Pubmed
Khundmiri,
Parathyroid hormone regulation of type II sodium-phosphate cotransporters is dependent on an A kinase anchoring protein.
2003,
Pubmed
Kimura,
Protein phosphatase 2A interacts with the Na,K-ATPase and modulates its trafficking by inhibition of its association with arrestin.
2011,
Pubmed
Konstas,
cAMP-dependent activation of CFTR inhibits the epithelial sodium channel (ENaC) without affecting its surface expression.
2003,
Pubmed
,
Xenbase
König,
The cystic fibrosis transmembrane conductance regulator (CFTR) inhibits ENaC through an increase in the intracellular Cl- concentration.
2001,
Pubmed
,
Xenbase
Lewarchik,
Regulation of CFTR trafficking by its R domain.
2008,
Pubmed
,
Xenbase
Lim,
Alpha4 integrins are type I cAMP-dependent protein kinase-anchoring proteins.
2007,
Pubmed
Ma,
Stimulatory and Inhibitory Functions of the R Domain on CFTR Chloride Channel.
2000,
Pubmed
McNicholas,
Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.
1996,
Pubmed
,
Xenbase
Michlig,
Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.
2005,
Pubmed
,
Xenbase
Murer,
Regulation of Na/Pi transporter in the proximal tubule.
2003,
Pubmed
Nagel,
CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes.
2005,
Pubmed
,
Xenbase
Nagel,
Non-specific activation of the epithelial sodium channel by the CFTR chloride channel.
2001,
Pubmed
,
Xenbase
Nijholt,
Modulation of hypothalamic NMDA receptor function by cyclic AMP-dependent protein kinase and phosphatases.
2000,
Pubmed
,
Xenbase
O'Keeffe,
Requirement for a kinase-specific chaperone pathway in the production of a Cdk9/cyclin T1 heterodimer responsible for P-TEFb-mediated tat stimulation of HIV-1 transcription.
2000,
Pubmed
Poppe,
Cyclic nucleotide analogs as probes of signaling pathways.
2008,
Pubmed
Prié,
Nephrolithiasis and osteoporosis associated with hypophosphatemia caused by mutations in the type 2a sodium-phosphate cotransporter.
2002,
Pubmed
,
Xenbase
Prié,
Genetic disorders of renal phosphate transport.
2010,
Pubmed
Schwake,
Surface expression and single channel properties of KCNQ2/KCNQ3, M-type K+ channels involved in epilepsy.
2000,
Pubmed
,
Xenbase
Shead,
Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human bone.
2007,
Pubmed
Shimkets,
The activity of the epithelial sodium channel is regulated by clathrin-mediated endocytosis.
1997,
Pubmed
,
Xenbase
Simard,
Homooligomeric and heterooligomeric associations between K+-Cl- cotransporter isoforms and between K+-Cl- and Na+-K+-Cl- cotransporters.
2007,
Pubmed
,
Xenbase
Song,
Airway surface liquid depth measured in ex vivo fragments of pig and human trachea: dependence on Na+ and Cl- channel function.
2009,
Pubmed
Street,
Analysis of bone mineral density and turnover in patients with cystic fibrosis: associations between the IGF system and inflammatory cytokines.
2006,
Pubmed
Suaud,
Genistein restores functional interactions between Delta F508-CFTR and ENaC in Xenopus oocytes.
2002,
Pubmed
,
Xenbase
Suaud,
Regulatory interactions of N1303K-CFTR and ENaC in Xenopus oocytes: evidence that chloride transport is not necessary for inhibition of ENaC.
2007,
Pubmed
,
Xenbase
Taddei,
Altered channel gating mechanism for CFTR inhibition by a high-affinity thiazolidinone blocker.
2004,
Pubmed
Wang,
Slc26a6 regulates CFTR activity in vivo to determine pancreatic duct HCO3- secretion: relevance to cystic fibrosis.
2006,
Pubmed
Weber,
Functional integrity of the vesicle transporting machinery is required for complete activation of cFTR expressed in xenopus laevis oocytes.
2001,
Pubmed
,
Xenbase
Xie,
A short segment of the R domain of cystic fibrosis transmembrane conductance regulator contains channel stimulatory and inhibitory activities that are separable by sequence modification.
2002,
Pubmed
Yan,
Cystic fibrosis transmembrane conductance regulator differentially regulates human and mouse epithelial sodium channels in Xenopus oocytes.
2004,
Pubmed
,
Xenbase
Yang,
Stimulation of the epithelial sodium channel (ENaC) by cAMP involves putative ERK phosphorylation sites in the C termini of the channel's beta- and gamma-subunit.
2006,
Pubmed
,
Xenbase