Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We demonstrated previously that Calu-3 airway epithelial cells sense adenosine on their luminal surface through adenosine A2B receptors coupled to adenylyl cyclase. Occupancy of these receptors leads to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel through protein kinase A (PKA) anchored at the apical membrane. Because luminal A2B receptor activation does not raise total cellular cAMP levels, we hypothesized that activation of phosphodiesterases (PDEs) confines cAMP generated by apical A2B receptors to a microdomain that includes the CFTR channel. Using reverse transcription-PCR, Western blotting, and activity measurements, PDE4D was identified as the major PDE species in airway epithelia. Consistent with these results, inhibitors of PDE4, but not PDE3, selectively abolished the lateral confinement of cAMP signaling in apical membrane patches during cell-attached recordings. Furthermore, stimulation of the CFTR in excised apical patches by rolipram and RS25344 indicated that PDE4 is localized in close proximity to the CFTR channel. Indeed, immunohistochemistry of human airway sections revealed that PDE4D is localized in the apical domain of the cell. PDE4 was activated after luminal adenosine exposure in a PKA-dependent manner. Because PDE4 activity is positively regulated by PKA, our results support a model whereby the PDE diffusion barrier is proportional to the degree of receptor stimulation. These findings underscore the concept that subcellular localization of individual PDE isozymes is an important mechanism for confining cAMP signaling to functional domains within cells.
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PMID:Phosphodiesterase 4D forms a cAMP diffusion barrier at the apical membrane of the airway epithelium. 1561 Oct 99

The assembly of the cystic fibrosis transmembrane regulator (CFTR) chloride channel is of interest from the broad perspective of understanding how ion channels and ABC transporters are formed as well as dealing with the mis-assembly of CFTR in cystic fibrosis. CFTR is functionally distinct from other ABC transporters because it permits bidirectional permeation of anions rather than vectorial transport of solutes. This adaptation of the ABC transporter structure can be rationalized by considering CFTR as a hydrolyzable-ligand-gated channel with cytoplasmic ATP as ligand. Channel gating is initiated by ligand binding when the protein is also phosphorylated by protein kinase A and made reversible by ligand hydrolysis. The two nucleotide-binding sites play different roles in channel activation. CFTR self-associates, possibly as a function of its activation, but most evidence, including the low-resolution three-dimensional structure, indicates that the channel is monomeric. Domain assembly and interaction within the monomer is critical in maturation, stability, and function of the protein. Disease-associated mutations, including the most common, DeltaF508, interfere with domain folding and association, which occur both co- and post-translationally. Intermolecular interactions of mature CFTR have been detected primarily with the N- and C-terminal tails, and these interactions have some impact not only on channel function but also on localization and processing within the cell. The biosynthetic processing of the nascent polypeptide leading to channel assembly involves transient interactions with numerous chaperones and enzymes on both sides of the endoplasmic reticulum membrane.
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PMID:Assembly of functional CFTR chloride channels. 1570 75

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed DeltaR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the DeltaR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal DeltaR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of DeltaR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and DeltaR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the DeltaR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.
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PMID:CFTR gating I: Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (DeltaR-CFTR). 1576 95

Single nucleotide polymorphisms (SNPs) in genes encoding or influencing renal sodium transport systems were investigated as potential predictors of blood pressure (BP) response to a thiazide diuretic. A sample of 585 adults with essential hypertension (30 to 59.9 years of age; 50% blacks; 47% women) were treated with hydrochlorothiazide for 4 weeks (25 mg daily, orally) to determine office BP responses. Ambulatory BP responses were measured in a subset of 228 subjects. After adjustment for ethnicity, sex, age, and waist-to-hip ratio, 3 SNPs in WNK1 (rs2107614, rs2277869, and rs1159744), encoding a lysine-deficient protein kinase that regulates thiazide-sensitive sodium-potassium cotransport, made statistically significant contributions to predicting ambulatory BP responses, accounting for 2% to 4% of variation in systolic and diastolic responses (P<0.05). SNPs in the beta2-adrenoceptor (rs2400707) and the epithelial sodium channel gamma-subunit (rs5723 and rs5729) were associated with similar magnitude of variation in ambulatory systolic BP response (P=0.028) or office diastolic BP response (P<0.05), respectively. However, SNPs evaluated in the furosemide-sensitive sodium-potassium chloride cotransporter, potassium inwardly rectifying channel, chloride channel, thiazide-sensitive sodium chloride cotransporter, epithelial sodium channel beta-subunit, and the mineralocorticoid receptor were not associated with significant variation in ambulatory or office BP responses. Polymorphisms in genes regulating renal sodium transport, in particular WNK1, predict interindividual differences in antihypertensive responses to hydrochlorothiazide.
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PMID:WNK1 kinase polymorphism and blood pressure response to a thiazide diuretic. 1617 12

The soy phytoestrogen genistein is a potent vasorelaxant, but its mechanism of action is poorly understood. Here, we used endothelium-denuded rat aorta to investigate the role of the cyclic AMP(cAMP)-activated, cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, and its associated Na-K-Cl cotransporter NKCC1. Isolated, endothelium-denuded rat aorta was contracted with phenylephrine 1 microM, and the vasorelaxant responses to genistein were investigated under conditions where CFTR was inhibited by DPC (diphenylamine-2-carboxylic acid) or glibenclamide (n=6 for compound). Both compounds fully antagonized the vasorelaxant responses to genistein, with IC50=57+/-18 microM and 42+/-11 microM for DPC and glibenclamide respectively. H-89, a selective protein kinase A (PKA) inhibitor, blocked the vasorelaxant responses to genistein. Finally, the NKCC1 inhibitor, bumetanide fully antagonized the vasorelaxant responses to genistein against phenylephrine- or KCl-induced contractions, with IC50=2.0+/-0.2 microM and 1.6+/-0.5 microM, respectively (n=6 for condition). These results strongly suggest that CFTR opening is involved in the vasorelaxant action of genistein, and that cAMP-dependent CFTR phosphorylation and chloride entry via the NKCC1 cotransporter are required for genistein action.
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PMID:Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and Na-K-Cl cotransporter NKCC1 isoform mediate the vasorelaxant action of genistein in isolated rat aorta. 1685 73

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase-dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing.
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PMID:The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics. 1696 75

The human ATP-binding cassette (ABC) protein CFTR (cystic fibrosis transmembrane conductance regulator) is a chloride channel, whose dysfunction causes cystic fibrosis. To gain structural insight into the dynamic interaction between CFTR's nucleotide-binding domains (NBDs) proposed to underlie channel gating, we introduced target cysteines into the NBDs, expressed the channels in Xenopus oocytes, and used in vivo sulfhydryl-specific crosslinking to directly examine the cysteines' proximity. We tested five cysteine pairs, each comprising one introduced cysteine in the NH(2)-terminal NBD1 and another in the COOH-terminal NBD2. Identification of crosslinked product was facilitated by co-expression of NH(2)-terminal and COOH-terminal CFTR half channels each containing one NBD. The COOH-terminal half channel lacked all native cysteines. None of CFTR's 18 native cysteines was found essential for wild type-like, phosphorylation- and ATP-dependent, channel gating. The observed crosslinks demonstrate that NBD1 and NBD2 interact in a head-to-tail configuration analogous to that in homodimeric crystal structures of nucleotide-bound prokaryotic NBDs. CFTR phosphorylation by PKA strongly promoted both crosslinking and opening of the split channels, firmly linking head-to-tail NBD1-NBD2 association to channel opening.
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PMID:In vivo phosphorylation of CFTR promotes formation of a nucleotide-binding domain heterodimer. 1703 51

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cyclic adenosine monophosphate (cAMP)-dependent chloride channel located mainly at the apical membrane of epithelial cells. In myocytes of pulmonary arteries, numerous chloride channels have been identified and described, but not the CFTR. Thus the presence and function of the CFTR was investigated in rat intrapulmonary arteries. CFTR expression, localisation and function were analysed in cultured smooth muscle cells using Reverse transcriptase (RT)-PCR and immunoprecipitation followed by protein kinase A phosphorylation, immunolocalisation and an iodide efflux assay, respectively. The role of the CFTR in pulmonary vasoreactivity was determined in arterial rings using an organ bath system. RT-PCR and immunoprecipitation analyses, as well as the immunolocalisation study, revealed the expression of CFTR gene transcripts and protein. The iodide efflux assay showed the existence of functional cAMP-, calcium- and volume-dependent chloride channels. Furthermore, the following effects were found: 1) inhibition of forskolin/genistein-activated iodide efflux by glibenclamide, diphenylamine-2-carboxylic acid and CFTR-specific inhibitor (CFTR(inh))-172; 2) activation of iodide efflux by the benzoquinolizinium derivative CFTR activators MPB-07 and MPB-91; and 3) inhibition of MPB-dependent efflux by CFTR(inh)-172. Finally, CFTR activators induced concentration-dependent vasorelaxation in rings preconstricted with phenylephrine, in the presence or absence of endothelium. The present results are the first to reveal functional cyclic adenosine monophosphate-regulated cystic fibrosis transmembrane conductance regulator contributing to endothelium-independent vasorelaxation in rat intrapulmonary arterial myocytes.
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PMID:Expression and function of cystic fibrosis transmembrane conductance regulator in rat intrapulmonary arteries. 1759 72

Recent studies have suggested a role for a chloride current in the modulation of pacemaker potentials generated by interstitial cells of Cajal. Patch-clamp recordings were made from inside-out patches of cultured interstitial cells of Cajal from the murine small intestine. The majority of patches were quiescent immediately after excision, but in some patches currents activated spontaneously after a period of 10 min to 1 h. Currents could also be activated by strongly polarizing the patch. It was found that the currents activated in both cases included a chloride channel. This channel could also be activated by ATP and the catalytic subunit of protein kinase A. The channel had conductance states (+/-SD) of 53 +/- 25.35, 126 +/- 21.44, 180 +/- 12.57 and 211 +/- 8.86 pS. It was outwardly rectifying (as a function of open probability) and deactivated (i.e., gave a tail current) but showed no inactivation. The permeability sequence of the channel was I(-)>>Br(-)>or=Cl(-)>Asp(-). It was unaffected in magnitude or rectification by changing the free Ca2+ concentration of the bath between <10 nM, 100 nM (control) and 2 mM .
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PMID:An outwardly rectifying and deactivating chloride channel expressed by interstitial cells of cajal from the murine small intestine. 1827 33

Cystic fibrosis (CF) is characterised by impaired epithelial ion transport and is caused by mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/PKA and ATP-regulated chloride channel. We recently demonstrated a cAMP/PKA/calcineurin (CnA)-driven association between annexin 2 (anx 2), its cognate partner -S100A10 and cell surface CFTR. The complex is required for CFTR and outwardly rectifying chloride channel function in epithelia. Since the cAMP/PKA-induced Cl(-) current is absent in CF epithelia, we hypothesized that the anx 2-S100A10/CFTR complex may be defective in CFBE41o cells expressing the commonest F508del-CFTR (DeltaF-CFTR) mutation. Here, we demonstrate that, despite the presence of cell surface DeltaF-CFTR, cAMP/PKA fails to induce anx 2-S100A10/CFTR complex formation in CFBE41o- cells homozygous for F508del-CFTR. Mechanistically, PKA-dependent serine phosphorylation of CnA, CnA-anx 2 complex formation and CnA-dependent dephosphorylation of anx 2 are all defective in CFBE41o- cells. Immunohistochemical analysis confirms an abnormal cellular distribution of anx 2 in human and CF mouse epithelia. Thus, we demonstrate that cAMP/PKA/CnA signaling pathway is defective in CF cells and suggest that loss of anx 2-S100A10/CFTR complex formation may contribute to defective cAMP/PKA-dependent CFTR channel function.
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PMID:Defective formation of PKA/CnA-dependent annexin 2-S100A10/CFTR complex in DeltaF508 cystic fibrosis cells. 1834 74


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