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)

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)-activated chloride channel that is localized to the plasma membrane and endosomal compartment. Endosomal targeting of CFTR is attributed to the Tyr(1424)-based internalization signal, identified in the C-terminal tail of the channel. Mutation of the Tyr(1424) residue could partly inhibit the endocytosis of CFTR and its association with the adapter protein AP-2. To reveal additional endosomal targeting signals, site-directed mutagenesis of both a chimaera, composed of a truncated form of interleukin 2 receptor alpha chain (TacT) and the C-terminal tail of CFTR (Ct), and the full-length CFTR was performed. Morphological and functional assays revealed the presence of multiple internalization motifs at the C-terminus, consisting of a phenylalanine-based motif (Phe(1413)) and a bipartite endocytic signal, comprising a tyrosine (Tyr(1424)) and a di-Leu-based (Leu(1430)-Leu) motif. Whereas the replacement of any one of the three internalization motifs with alanine prevented the endocytosis of the TacT-Ct chimaera, mutagenesis of Phe(1413)-Leu impaired the biosynthetic processing of CFTR, indicating that Phe(1413) is indispensable for the native structure of CFTR. In contrast, replacement of Leu(1430)-Leu- and Tyr(1424)-based signals with alanine increased the cell-surface density of both the chimaeras and CFTR in an additive manner. These results suggest that the internalization of CFTR is regulated by multiple endocytic sorting signals.
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PMID:Multiple endocytic signals in the C-terminal tail of the cystic fibrosis transmembrane conductance regulator. 1123 60

After phosphorylation by protein kinase A, gating of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is regulated by the interaction of ATP with its nucleotide binding domains (NBDs). Models of this gating regulation have proposed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed in Nagel, G. (1999) Biochim. Biophys. Acta 1461, 263-274). However, as yet there has been little biochemical confirmation of the predictions of these models. We have employed photoaffinity labeling with 8-azido-ATP, which supports channel gating as effectively as ATP to evaluate interactions with each NBD in intact membrane-bound CFTR. Mutagenesis of Walker A lysine residues crucial for azido-ATP hydrolysis to generate the azido-ADP that is trapped by vanadate indicated a greater role of NBD1 than NBD2. Separation of the domains by limited trypsin digestion and enrichment by immunoprecipitation confirmed greater and more stable nucleotide trapping at NBD1. This asymmetry of the two domains in interactions with nucleotides was reflected most emphatically in the response to the nonhydrolyzable ATP analogue, 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), which in the gating models was proposed to bind with high affinity to NBD2 causing inhibition of ATP hydrolysis there postulated to drive channel closing. Instead we found a strong competitive inhibition of nucleotide hydrolysis and trapping at NBD1 and a simultaneous enhancement at NBD2. This argues strongly that AMP-PNP does not inhibit ATP hydrolysis at NBD2 and thereby questions the relevance of hydrolysis at that domain to channel closing.
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PMID:Differential interactions of nucleotides at the two nucleotide binding domains of the cystic fibrosis transmembrane conductance regulator. 1127 83

We have studied the regulation of Ca(2+)-dependent chloride (Cl(Ca)) channels in a human pancreatoma epithelial cell line (CFPAC-1), which does not express functional cAMP-dependent cystic fibrosis transmembrane conductance regulator chloride channels. In cell-free patches from these cells, physiological Ca(2+) concentrations activated a single class of 1-picosiemens Cl(-)-selective channels. The same channels were also stimulated by a purified type II calmodulin-dependent protein kinase (CaMKII), and in cell-attached patches by purinergic agonists. In whole-cell recordings, both Ca(2+)- and CaMKII-dependent mechanisms contributed to chloride channel stimulation by Ca(2+), but the CaMKII-dependent pathway was selectively inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)). This inhibitory effect of Ins(3,4,5,6)P(4) on Cl(Ca) channel stimulation by CaMKII was reduced by raising [Ca(2+)] and prevented by inhibition of protein phosphatase activity with 100 nm okadaic acid. These data provide a new context for understanding the physiological relevance of Ins(3,4,5,6)P(4) in the longer term regulation of Ca(2+)-dependent Cl(-) fluxes in epithelial cells.
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PMID:Regulation of a human chloride channel. a paradigm for integrating input from calcium, type ii calmodulin-dependent protein kinase, and inositol 3,4,5,6-tetrakisphosphate. 1127 75

Guanylin and uroguanylin are short peptides homologous to heat-stable enterotoxins of Escherichia coli and other enteric bacteria. Guanylin and uroguanylin are synthetized from the respective prepropeptides mainly in gastrointestinal mucosa and are secreted both into intestinal lumen and into the blood. Luminally secreted peptides stimulate chloride and bicarbonate secretion in the intestine through the mechanism involving guanylate cyclase C receptor, cyclic GMP, protein kinase G and cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Bacterial enterotoxins, which have greater potency than endogenous peptides, induce excessive fluid secretion into intestinal lumen leading to secretory diarhea. Uroguanylin is expressed mainly in enterochromaffin cells of duodenum and proximal small intestine whereas guanylin is abundant in goblet cells of colonic epithelium. Uroguanylin and guanylin increase urinary sodium and potassium excretion both as circulating hormones and as paracrine mediators produced within the kidney. Uroguanylin functions as "intestinal natriuretic hormone" which is secreted in response to oral sodium loading and maintains sodium balance during postprandial period. Plasma and urinary concentrations of guanylin and uroguanylin increase in renal failure and heart failure. Guanylin peptides possess antiproliferative activity in intestinal cells culture and their expression decreases in colonic carcinoma indicating that their deficiency may contribute to the pathogenesis of this disease.
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PMID:Guanylin and related peptides. 1159 56

Epithelia of humans and mice with cystic fibrosis are unable to secrete chloride in response to a chloride gradient or to cAMP-elevating agents. Bioelectrical properties measured using the nasal transepithelial potential difference (TEPD) assay are believed to reflect these cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride transport defects. Although the response to forskolin is CFTR mediated, the mechanisms responsible for the response to a chloride gradient are unknown. TEPD measurements performed on inbred mice were used to compare the responses to low chloride and forskolin in vivo. Both responses show little correlation between or within inbred strains of mice, suggesting they are mediated through partially distinct mechanisms. In addition, these responses were assayed in the presence of several chloride channel inhibitors, including DIDS, diphenylamine-2-carboxylate, glibenclamide, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and a protein kinase A inhibitor, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). The responses to low chloride and forskolin demonstrate significantly different pharmacological profiles to both DIDS and Rp-cAMPS, indicating that channels in addition to CFTR contribute to the low chloride response.
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PMID:Examining basal chloride transport using the nasal potential difference response in a murine model. 1159 9

1. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PKA) phosphorylation of its R domain and by ATP binding at its nucleotide-binding domains (NBDs). Here we investigated the functional role of a cluster of acidic residues in the amino terminal tail (N-tail) that also modulate CFTR channel gating by an unknown mechanism. 2. A disease-associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP -activating cocktail than wild-type CFTR. 3. In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bursts. 4. Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation. 5. Conversely, the N-tail triple mutant exhibited a markedly inhibited response to AMP-PNP, a poorly hydrolysable ATP analogue that can nearly lock open the wild-type channel. The N-tail mutant had both a slower response to AMP-PNP (activation half-time of 140 +/- 20 s vs. 21 +/- 4 s for wild type) and a lower steady-state open probability following AMP-PNP addition (0.68 +/- 0.08 vs. 0.92 +/- 0.03 for wild type). 6. Introducing the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts. 7. We propose that this cluster of acidic residues modulates the stability of CFTR channel openings at a step that is downstream of ATP binding and upstream of ATP hydrolysis, probably at NBD2.
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PMID:A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating. 1160 Jun 81

1. We identified the ethacrynic-acid derivative DCPIB as a potent inhibitor of I(Cl,swell), which blocks native I(Cl,swell) of calf bovine pulmonary artery endothelial (CPAE) cells with an IC(50) of 4.1 microM. Similarly, 10 microM DCPIB almost completely inhibited the swelling-induced chloride conductance in Xenopus oocytes and in guinea-pig atrial cardiomyocytes. Block of I(Cl,swell) by DCPIB was fully reversible and voltage independent. 2. DCPIB (10 microM) showed selectivity for I(Cl,swell) and had no significant inhibitory effects on I(Cl,Ca) in CPAE cells, on chloride currents elicited by several members of the CLC-chloride channel family or on the human cystic fibrosis transmembrane conductance regulator (hCFTR) after heterologous expression in Xenopus oocytes. DCPIB (10 microM) also showed no significant inhibition of several native anion and cation currents of guinea pig heart like I(Cl,PKA), I(Kr), I(Ks), I(K1), I(Na) and I(Ca). 3. In all atrial cardiomyocytes (n=7), osmotic swelling produced an increase in chloride current and a strong shortening of the action potential duration (APD). Both swelling-induced chloride conductance and AP shortening were inhibited by treatment of swollen cells with DCPIB (10 microM). In agreement with the selectivity for I(Cl,swell), DCPIB did not affect atrial APD under isoosmotic conditions. 4. Preincubation of atrial cardiomyocytes with DCPIB (10 microM) completely prevented both the swelling-induced chloride currents and the AP shortening but not the hypotonic cell swelling. 5. We conclude that swelling-induced AP shortening in isolated atrial cells is mainly caused by activation of I(Cl,swell). DCPIB therefore is a valuable pharmacological tool to study the role of I(Cl,swell) in cardiac excitability under pathophysiological conditions leading to cell swelling.
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PMID:DCPIB is a novel selective blocker of I(Cl,swell) and prevents swelling-induced shortening of guinea-pig atrial action potential duration. 1172 53

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR is a chloride channel whose activity requires protein kinase A-dependent phosphorylation of an intracellular regulatory domain (R-domain) and ATP hydrolysis at the nucleotide-binding domains (NBDs). To identify potential sites of domain-domain interaction within CFTR, we expressed, purified, and refolded histidine (His)- and glutathione-S-transferase (GST)-tagged cytoplasmic domains of CFTR. ATP-binding to his-NBD1 and his-NBD2 was demonstrated by measuring tryptophan fluorescence quenching. Tryptic digestion of in vitro phosphorylated his-NBD1-R and in situ phosphorylated CFTR generated the same phosphopeptides. An interaction between NBD1-R and NBD2 was assayed by tryptophan fluorescence quenching. Binding among all pairwise combinations of R-domain, NBD1, and NBD2 was demonstrated with an overlay assay. To identify specific sites of interaction between domains of CFTR, an overlay assay was used to probe an overlapping peptide library spanning all intracellular regions of CFTR with his-NBD1, his-NBD2, and GST-R-domain. By mapping peptides from NBD1 and NBD2 that bound to other intracellular domains onto crystal structures for HisP, MalK, and Rad50, probable sites of interaction between NBD1 and NBD2 were identified. Our data support a model where NBDs form dimers with the ATP-binding sites at the domain-domain interface.
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PMID:Domain-domain associations in cystic fibrosis transmembrane conductance regulator. 1194 May 32

The regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) contains consensus phosphorylation sites for cAMP-dependent protein kinase (PKA) that are the basis for physiological regulation of the CFTR chloride channel. A short peptide segment in the R domain with a net negative charge of B9 (amino acids 817-838, NEG2) and predicted helical tendency is shown to play a critical role in CFTR chloride channel function. Deletion of NEG2 from CFTR completely eliminates the PKA dependence of channel activity. Exogenous NEG2 peptide interacts with CFTR to exert both stimulatory and inhibitory effects on the channel function. The NEG2 peptide with sequence scrambled to remove helical tendencies also inhibits channel function, but does not stimulate. Similar results are found for a NEG2 peptide whose helical structure is disrupted by a proline residue. When six of the negatively charged carboxylic acid residues are replaced by their cognate amides, reducing net negative charge to B3, but increasing helical propensity as assessed by circular dichroism, the peptide stimulates CFTR channel function, but does not inhibit. We speculate that the NEG2 region interacts with other cytosolic domains of CFTR to control opening and closing transitions of the chloride channel.
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PMID: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. 1195 Aug 44

We studied gamma-aminobutyric acid (GABA)-mediated currents in short-term cultured dorsal unpaired median (DUM) neurons of cockroach Periplaneta americana using the whole cell patch-clamp technique in symmetrical chloride solutions. All DUM neurons voltage-clamped at -50 mV displayed inward currents (I(GABA)) when 10(-4) M of GABA was applied by pneumatic pressure-ejection pulses. The semi-logarithmic curve of I(GABA) amplitude versus the ejection time yielded a Hill coefficient of 4.0. I(GABA) was chloride (Cl-) because the reversal potential given by the current-voltage (I-V) curve varied according to the value predicted by the Nernst equation for Cl- dependence. In addition, I(GABA) was almost completely blocked by bath application of the chloride channel blockers picrotoxin (PTX) or 3,3-bis(trifluoromethyl)bicyclo-[2,2,1]heptane-2,2-diacarbonitrile (BIDN). The I-V curve for I(GABA) displayed a unexpected biphasic aspect and was best fitted by two linear regressions giving two slope conductances of 35.6 +/- 2.1 and 80.9 +/- 4.1 nS for potentials ranging from 0 to -30 and -30 to -70 mV, respectively. At -50 mV, the current amplitude was decreased by cadmium chloride (CdCl2, 10(-3) M) and calcium-free solution. The semi-logarithmic curve for CdCl2-resistant I(GABA) gave a Hill coefficient of 2.4. Hyperpolarizing voltage step from -50 to -80 mV was known to increase calcium influx through calcium-resting channels. According to this protocol, a significant increase of I(GABA) amplitude was observed. However, this effect was never obtained when the same protocol was applied on cell body pretreated with CdCl2. When the calmodulin blocker N-(6-aminohexyl)-5-chloro-1-naphtalene-sulfonamide or the calcium-calmodulin-dependent protein kinase blocker 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) was added in the pipette solution, I(GABA) amplitude was decreased. Pressure ejection application of the cis-4-aminocrotonic acid (CACA) on DUM neuron cell body held at -50 mV, evoked a Cl- inward current which was insensitive to CdCl2. The Hill plot yielded a Hill coefficient of 2.3, and the I-V curve was always linear in the negative potential range with a slope conductance of 32.4 +/- 1.1 nS. These results, similar to those obtained with GABA in the presence of CdCl2 and KN-62, indicated that CACA activated one subtype of GABA receptor. Our study demonstrated that at least two distinct subtypes of Cl--dependent GABA receptors were expressed in DUM neurons, one of which is regulated by an intracellular Ca2+-dependent mechanism via a calcium-dependent protein kinase. The consequences of the modulatory action of Ca2+ in GABA receptors function and their sensitivity to insecticide are discussed.
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PMID:Ca2+/calmodulin-dependent protein kinase regulates GABA-activated Cl- current in cockroach dorsal unpaired median neurons. 1203


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