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)

Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type I alpha cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl- channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl-channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl- channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATP gamma S (adenosine 5'-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKI alpha, is a potential activator of chloride transport in CFTR-expressing cell types.
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PMID:Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II. 759 87

Stimulation of the beta-adrenoceptor activates a time-independent Cl- conductance that is known to be regulated via phosphorylation by cAMP-dependent protein kinase in guinea pig ventricular myocytes. Since epithelial cystic fibrosis transmembrane conductance regulator Cl- channels are known to be sensitive to an antidiabetic sulfonylurea, glibenclamide, we tested whether the drug modulates cardiac cAMP-activated Cl- conductance. Bath application of isoproterenol (1 mumol/L, n = 11) or forskolin (1 mumol/L, n = 17) or the intracellular application of cAMP (1 mmol/L, n = 9) activated whole-cell Cl- currents recorded from single myocytes at 36 degrees C. External glibenclamide (> or = 10 mumol/L, n = 26) inhibited the Cl- current induced by either of the stimulants in a concentration-dependent manner. The half-maximal inhibition concentration (IC50) of glibenclamide and the Hill coefficient were 24.5 to 37.9 mumol/L and 1.6 to 2.2, respectively. During current-clamp experiments, forskolin was found to shorten the action potential significantly (250 +/- 45 to 201 +/- 52 milliseconds, P < .05) in 7 of 11 cells tested. Glibenclamide antagonized the forskolin-induced shortening (to 243 +/- 54 milliseconds, n = 7, P < .05). Intracellular administration of sodium orthovanadate (0.5 to approximately 1 mmol/L, n = 6) brought about persistent activation of Cl- current after brief bath application of forskolin. This Cl- current was not affected by H-89 (100 mumol/L, n = 3), a specific inhibitor of cAMP-dependent protein kinase, and was suppressed by glibenclamide similarly, with an IC50 of 29.7 mumol/L.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Glibenclamide, an ATP-sensitive K+ channel blocker, inhibits cardiac cAMP-activated Cl- conductance. 761 25

In cystic fibrosis (CF), numerous epithelial cell functions are abnormal, including Cl- conductance, sodium absorption, mucin sulphation and enzyme secretion. Although the CF gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), functions as a small linear Cl- channel, it is difficult to attribute such pleiotropic disease manifestations solely to a defect in Cl- conductance. This has led to speculation that CFTR regulates the activity of other proteins. One possible example is the protein kinase A activation of outward rectifying Cl- channels (ORCC), which is defective in membrane patches excised from CF cells. Whether CFTR regulates the activity of an independent anion channel is debatable, because ORCC occur exclusively in excised membrane patches and could be an excision-induced molecular derivative of CFTR. 'Knockout' mice that lack CFTR provide a means to define the relationship between CFTR and ORCC. Here we report that ORCC are present in CFTR(-/-) mouse nasal epithelial cells and thus cannot be a derivative of the CFTR molecule. Also ORCC were regulated by protein kinase A in membrane patches from normal but not CFTR(-/-) cells. These observations are the first, to our knowledge definitive demonstration that CFTR regulates the activity of another protein.
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PMID:CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship. 768 73

The regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator (CFTR) is phosphorylated by protein kinase A and protein kinase C (PKC) in vivo (Picciotto, M. R., Cohn, J. A., Bertuzzi, G., Greengard, P., and Nairn, A. C. (1992) J. Biol. Chem. 267, 12742-12752), but so far the functional effect of the PKC-dependent phosphorylation has not been clarified. We investigated the effect of PKC on the CFTR-mediated Cl- transport by treating with phorbol 12-myristate 13-acetate (PMA), the cell line C127i stably expressing CFTR wild type (C127 CFTRw/t), or CFTR bearing the most common mutation deltaF508 (C127 CFTRdF508). We show that PMA activates Cl- efflux in C127 CFTRw/t, but not in C127 CFTRdF508 and C127i. The PMA-dependent activation of CFTR is not mediated by increase of intracellular [cAMP] and is not the result of a primary activation of a K+ conductive pathway. These results strongly suggest that PKC activates directly CFTR-mediated Cl- transport.
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PMID:Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene. 768 79

Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.
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PMID:Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain. 769 Jul 53

The past decade of research in cystic fibrosis has produced a wealth of information about the underlying defect responsible for the disease. The initial finding that the physiological disturbance in CF is one of abnormal electrolyte transport across epithelial tissues led to the elucidation of a pathway in which epithelial chloride transport is normally elicited in response to beta-adrenergic stimuli and involves the second messenger cAMP to activate protein kinase A. While that pathway was being described, work on the genetic front was concurrently providing information about the genomic location of the gene causing CF, which ultimately led to the identification and cloning of the gene encoding the cystic fibrosis transmembrane conductance regulator. The cloned CFTR gene provided a powerful reagent to use in the next generation of cell physiology experiments, in which it was determined that CFTR is not only the substrate of PKA phosphorylation, a step previously determined to be in the activation pathway of the chloride channel, but is in fact a cAMP-dependent chloride conducting channel itself. Further analysis of the gene has shown that although there is a single mutation that accounts for most of CF, there are well over 200 other lesions within the gene that can cause disease as well. Identification of these mutations has provided information into the normal function of CFTR by studying these variants in heterologous expression systems. As a result, the molecular mechanism of CFTR function is beginning to unfold, as well as the mechanism by which particular mutations impair that function. From a clinical perspective, the research brings optimism from two directions. First, understanding how disease-causing mutations impair function may culminate in pharmacologic approaches that can restore function to some of these mutants. Second, treating the disease at the level of the gene appears to be a realistic goal: Gene transfer experiments in cultured CF cells have shown that the procedure will restore cAMP-dependent chloride conductance to the cells, laying the groundwork for somatic cell gene therapy as a feasible treatment for CF. Currently, work is rapidly progressing in developing delivery systems for this purpose. Finally, animal models that should not only aid in understanding the physiology of electrolyte transport in epithelia but should serve as indicators for tests of therapeutic approaches to treating CF are being developed, either by pharmacological means or by gene delivery protocols.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Molecular biology of cystic fibrosis. 769 8

Findings outlined here support a complex model for the regulation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel gating that incorporates incremental protein kinase A (PKA) phosphorylation of CFTR at multiple sites which, in turn, differentially control the activity of CFTR's two nucleotide-binding domains (NBDs). The NBDs are functionally distinct: only one can respond to the non-hydrolyzable ATP analogue AMP-PNP, and then only after ATP has acted at the other. Moreover, the nature of the responses to AMP-PNP, and to the inorganic phosphate analogue orthovanadate, argues that ATP hydrolysis normally occurs at both NBDs, at one to initiate channel opening and at the other to initiate closing.
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PMID:Regulation of CFTR channel gating. 775 25

Whole cell and single-channel patch-clamp techniques were used to identify and characterize the Cl- currents responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the rectal gland of the spiny dogfish (Squalus acanthias). During whole cell recordings, in cultured rectal gland cells forskolin (10 microM) and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (400 microM) stimulated a 28-fold increase in Cl- conductance (n = 10). This cAMP-activated conductance pathway had a linear current-voltage (I-V) relationship that was time and voltage independent. Substitution of 235 meq Cl- with I- in the bath inhibited the cAMP-activated current at both positive and negative voltages (64%). Glibenclamide (60 microM) abolished the cAMP-stimulated current, and its effect was irreversible (n = 3). During cell-attached recording, increased cellular cAMP activated single Cl- channels in nine previously quiet patches. These channels had a linear I-V relationship with an average single-channel conductance of 5.1 +/- 0.2 pS (n = 6). Similar properties were observed in excised inside-out patches, permitting further characterization of the single-channel properties. Excised quiescent patches could be activated by the addition of ATP and protein kinase A. Replacing bath Cl- with I- inhibited both inward and outward currents (n = 3). In three inside-out patches, glibenclamide (300 microM) reversibly reduced open probability by 74%, with no effect on single-channel current amplitude. Similar results were obtained in four outside-out recordings. These results suggest that increased cellular cAMP in dogfish rectal gland activates a small linear Cl- channel that resembles human cystic fibrosis transmembrane conductance regulator in its biophysical and pharmacological properties.
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PMID:cAMP-activated Cl- channels in primary cultures of spiny dogfish (Squalus acanthias) rectal gland. 784 Jan 62

Using the patch clamp technique on the apical membrane of primary cultures of rabbit distal bright convoluted tubule cells (DCTb), two types of Cl- channel were identified. A small channel of 9 pS was observed in 9% of the patches. Cells pretreated with 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or 5 microM forskolin increased the expression of Cl- channels by 26 and 37%, respectively. In cell-attached and excised inside-out patches, the current-voltage (I-V) relationships of the 9-pS channel were linear. In only 1 out of 47 active patches was the small-conductance Cl- channel still active 1 h after membrane excision. The addition of 0.1 microM of the catalytic subunit protein kinase A with 2 mM ATP to the cytoplasmic side restored channel activity in 8 out of 15 excised membrane patches. In 5 out of 467 patches of stimulated or nonstimulated cells, a larger Cl- conductance of 30 pS was also recorded. In excised inside-out patches this channel outwardly rectified and was activated by strong depolarization. In cultured DCTb cells, the small-conductance, cAMP-activated Cl- channel shares many properties with the cystic fibrosis transmembrane conductance regulator. Our results suggest that at least the small-conductance channel may participate in Cl- secretion across the apical membrane of DCTb in primary culture. This secretion may increase the rate of the apical Cl-/HCO3- exchange indirectly by enhancing the inwardly-directed Cl- gradient.
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PMID:Chloride channels in apical membrane of primary cultures of rabbit distal bright convoluted tubule. 818 86

Cystic fibrosis (CF) is a frequent autosomal recessive genetic disease. The isolation of the gene at the CF locus assigned to the long arm of chromosome 7 band q 31 and defining description of its protein named CFTR (cystic fibrosis transmembrane conductance regulator) promoted understanding the basic biochemical defect. Brief review of relevant literature demonstrates that glycoprotein CFTR is a chloride channel and is activated by a combination of phosphorylation by protein kinase A and binding of ATP. Most common mutation of CF gene, a deletion of the three nucleotides encoding phenylalanine (Delta F508) results in disturbance of chloride transport through membrane of epithelial cells involved in pathomechanism of CF. The way for gene therapy in CF is open, however therapeutic progress is noted on both pharmacologic arena and on the gene cure front. Recombinant vectors utilizing the adenovirus system with high efficiency of CFTR gene transfer to airway epithelium demonstrated in a rat model look promising. The use of retroviruses for CFTR transfer is also advanced mode of somatic gene therapy. An alternative approach suggesting the use of germ line cells is prerequisite of the development of the preimplantation/preconception genetic CF diagnosis. A number of safety and efficacy issues have to be addressed for all approaches before human trials can be implemented.
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PMID:[Gene therapy perspectives in cystic fibrosis]. 830 49


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