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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In many species the pancreatic duct epithelium secretes HCO3- ions at a concentration of around 140 mM by a mechanism that is only partially understood. We know that HCO3- uptake at the basolateral membrane is achieved by Na+-HCO3- cotransport and also by a H+-ATPase and Na+/H+ exchanger operating together with carbonic anhydrase. At the apical membrane, the secretion of moderate concentrations of HCO3- can be explained by the parallel activity of a Cl-/HCO3- exchanger and a Cl- conductance, either the cystic fibrosis transmembrane conductance regulator (CFTR) or a Ca2+-activated Cl- channel (CaCC). However, the sustained secretion of HCO3- into a HCO- -rich luminal fluid cannot be explained by conventional Cl-/HCO3- exchange. HCO3- efflux across the apical membrane is an electrogenic process that is facilitated by the depletion of intracellular Cl-, but it remains to be seen whether it is mediated predominantly by CFTR or by an electrogenic SLC26 anion exchanger.
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PMID:Mechanisms of bicarbonate secretion in the pancreatic duct. 1570 63

Mozambique tilapia Oreochromis mossambicus embryos were transferred from freshwater to seawater and vice versa, and short-term changes in the localization of three major ion transport proteins, Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR) were examined within mitochondrion-rich cells (MRCs) in the embryonic yolk-sac membrane. Triple-color immunofluorescence staining allowed us to classify MRCs into four types: type I, showing only basolateral Na+/K+-ATPase staining; type II, basolateral Na+/K+-ATPase and apical NKCC; type III, basolateral Na+/K+-ATPase and basolateral NKCC; type IV, basolateral Na+/K+-ATPase, basolateral NKCC and apical CFTR. In freshwater, type-I, type-II and type-III cells were observed. Following transfer from freshwater to seawater, type-IV cells appeared at 12 h and showed a remarkable increase in number between 24 h and 48 h, whereas type-III cells disappeared. When transferred from seawater back to freshwater, type-IV cells decreased and disappeared at 48 h, type-III cells increased, and type-II cells, which were not found in seawater, appeared at 12 h and increased in number thereafter. Type-I cells existed consistently irrespective of salinity changes. These results suggest that type I is an immature MRC, type II is a freshwater-type ion absorptive cell, type III is a dormant type-IV cell and/or an ion absorptive cell (with a different mechanism from type II), and type IV is a seawater-type ion secretory cell. The intracellular localization of the three ion transport proteins in type-IV cells is completely consistent with a widely accepted model for ion secretion by MRCs. A new model for ion absorption is proposed based on type-II cells possessing apical NKCC.
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PMID:Functional classification of mitochondrion-rich cells in euryhaline Mozambique tilapia (Oreochromis mossambicus) embryos, by means of triple immunofluorescence staining for Na+/K+-ATPase, Na+/K+/2Cl- cotransporter and CFTR anion channel. 1591 46

We have explored the molecular basis for differences in physiological function between the gills and opercular epithelium of the euryhaline killifish Fundulus heteroclitus. These tissues are functionally similar in seawater, but in freshwater the gills actively absorb Na+ but not Cl-, whereas the opercular epithelium actively absorbs Cl- but not Na+. These differences in freshwater physiology are likely due to differences in absolute levels of gene expression (measured using real-time PCR), as several proteins important for Na+ transport, namely Na+,H+-exchanger 2 (NHE2), carbonic anhydrase 2 (CA2), Na+,HCO3- cotransporter 1, and V-type H+-ATPase, were expressed at 3- to over 30-fold higher absolute levels in the gills. In gills, transfer from 10% seawater to freshwater increased the activity of Na+,K+-ATPase by twofold (from 12 h to 7 days), increased the expression of NHE2 (at 12 h) and CA2 (from 12 h to 7 days), and decreased the expression of NHE3 (from 12 h to 3 days). In opercular epithelium, NHE2 was not expressed; furthermore, Na+,K+-ATPase activity was unchanged after transfer to freshwater, CA2 mRNA levels decreased, and NHE3 levels increased. Consistent with their functional similarities in seawater, killifish gills and opercular epithelium expressed Na+,K+-ATPase alpha 1a, Na+,K+,2Cl- cotransporter 1 (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel and the signalling protein 14-3-3a at similar absolute levels. Furthermore, NKCC1 and CFTR were suppressed equally in each tissue after freshwater transfer, and 14-3-3a mRNA increased in both. These results provide insight into the mechanisms of ion transport by killifish gills and opercular epithelia, and demonstrate a potential molecular basis for the differences in physiological function between these two organs.
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PMID:Gene expression after freshwater transfer in gills and opercular epithelia of killifish: insight into divergent mechanisms of ion transport. 1600 May 41

The cystic fibrosis transmembrane conductance regulator (CFTR) is a channel/enzyme which mediates passive diffusion of chloride and bicarbonate through epithelial cell membranes. It is expressed in many cell types throughout the body, but in the airways it is found mainly in secretory serous cells of the submucosal glands. CFTR belongs to a large super-family of ATP binding cassette transporters that have two nucleotide binding domains with characteristic sequences or "motifs". Although most other ATP binding cassette transporters consume ATP to actively transport various substrates, in CFTR the interactions of ATP with nucleotide binding domains control opening and closing of the channel pore (i.e., channel gating). Recent high resolution structures of bacterial nucleotide binding domains combined with new biochemical and electrophysiological studies of CFTR itself have led to major advances in our understanding of CFTR gating. For example, it is now clear that the ATPase activity of CFTR is not strictly required for its channel activity. CFTR has at least two distinct gating modes; one dependent on hydrolysis and the other requiring only stable ATP binding. In this article we discuss a working hypothesis for CFTR that incorporates these recent findings and discuss some interesting implications of the paradigm shift for other aspects of CFTR function and dysfunction.
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PMID:Revisiting cystic fibrosis transmembrane conductance regulator structure and function. 1611 6

Cystic fibrosis (CF) is most commonly caused by deletion of Phe508 in the cystic fibrosis transmembrane conductance regulator protein (DeltaF508 CFTR). The misfolded DeltaF508 CFTR protein is retained in the endoplasmic reticulum (misprocessed mutant) and is rapidly degraded. Studies on misprocessed mutants of P-glycoprotein (P-gp), a sister protein of CFTR, however, have shown that specific substrates and modulators can act as specific chemical/pharmacological chaperones to rescue the protein. A major goal in CF research is the identification of compounds that can be used at low concentrations to rescue misprocessed CFTR mutants. Here, we show that a novel quinazoline derivative, 4-cyclohexyloxy-2-{1-[4-(4-methoxy-benzenesulfonyl)piperazin-1-yl]ethyl}quinazoline (CF(cor)-325), rescued DeltaF508 CFTR. Incubation of BHK cells stably expressing human DeltaF508 CFTR with 1-10 microM CF(cor)-325 resulted in maturation and delivery of a functional molecule to the cell surface as determined by the iodide efflux assay. The misprocessed CFTR mutants R258G, S945L, and H949Y were also rescued by CF(cor)-325 in either BHK or HEK 293 cells. CF(cor)-325 appeared to be specific for DeltaF508 CFTR because another quinazoline derivative, prazosin, did not rescue the misprocessed CFTR mutants. CF(cor)-325 could also rescue misprocessed mutants of P-gp. The compound was a P-gp inhibitor as it inhibited vinblastine-stimulated ATPase activity. P-gp-mediated vinblastine resistance was also reduced about 10-fold with 300 nM CF(cor)-325. These results show that CF(cor)-325 is a particularly important lead compound for treatment of CF because low concentrations can be used to rescue many misprocessed CFTR mutants.
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PMID:Rescue of DeltaF508 and other misprocessed CFTR mutants by a novel quinazoline compound. 1619 93

We have recently established a unique in vitro experimental model for mitochondrion-rich cell (MRC) research, a ;yolk-ball' incubation system, in which the yolk sac is separated from the embryonic body of Mozambique tilapia embryos and subjected to in vitro incubation. To evaluate the ion-transporting property of the yolk balls, we examined Cl- content and turnover in yolk balls incubated in freshwater and seawater for 48 h, and distribution patterns of three ion transporters, Na+/K+-ATPase, Na+/K+/2Cl- cotransporter (NKCC) and cystic fibrosis transmembrane conductance regulator (CFTR), in MRCs in the yolk-sac membrane. The Cl- turnover rate measured by whole-body influx of 36Cl- was about 60 times higher in yolk balls in seawater than in freshwater, while there was no essential difference in Cl- content between them. Na+/K+-ATPase-immunoreactive MRCs were larger in yolk balls from seawater than yolk balls from freshwater. Distribution patterns of ion-transporting proteins allowed us to classify MRCs in freshwater yolk balls into three types: cells showing only basolateral Na+/K+-ATPase, cells showing basolateral Na+/K+-ATPase and apical NKCC, and cells showing basolateral Na+/K+-ATPase and basolateral NKCC. The seawater yolk balls, on the other hand, were characterized by the appearance of MRCs possessing basolateral Na+/K+-ATPase, basolateral NKCC and apical CFTR. Those seawater-type MRCs were considered to secrete Cl- through the CFTR-positive apical opening to cope with diffusional Cl- influx. These findings indicate that the yolk balls preserve the Cl- transporting property of intact embryos, ensuring the propriety of the yolk ball as an in vitro experimental model for the yolk-sac membrane that contains MRCs.
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PMID:Chloride turnover and ion-transporting activities of yolk-sac preparations (yolk balls) separated from Mozambique tilapia embryos and incubated in freshwater and seawater. 1621 13

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.
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PMID:Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis. 1636 Dec 59

We examined the role of the cysteine string protein (Csp) in cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis in relation to another J-domain protein, Hdj-2, a recognized CFTR cochaperone. Increased expression of Csp produced a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR. Exogenous expression of Hdj-2 also increased CFTR band B, but unlike Csp, Hdj-2 increased band C as well. The Csp-induced block of CFTR maturation required Hsp70, because a J-domain mutant (H43Q) that interferes with the ability of Csp to stimulate Hsp70 ATPase activity relieved the Csp-induced block of CFTR maturation. Nevertheless, Csp H43Q still increased immature CFTR. Csp-induced band B CFTR was found adjacent to the nucleus, co-localizing with calnexin, and it remained detergent-soluble. These data indicate that Csp did not block CFTR maturation by promoting the aggregation or degradation of immature CFTR. Csp knockdown by RNA interference produced a 5-fold increase in mature CFTR and augmented cAMP-stimulated CFTR currents. Thus, the production of mature CFTR is inversely related to the expression level of Csp. Both Csp and Hdj-2 associated with the CFTR R-domain in vitro, and Hdj-2 binding was displaced by Csp, suggesting common interaction sites. Combined expression of Csp and Hdj-2 mimicked the effect of Csp alone, a block of CFTR maturation. But together, Csp and Hdj-2 produced additive increases in CFTR band B, and this did not depend on their interactions with Hsp70, consistent with direct chaperone actions of these proteins. Like Hdj-2, Csp reduced the aggregation of NBD1 in vitro in the absence of Hsp70. Our data suggest that both Csp and Hdj-2 facilitate the biosynthesis of immature CFTR, acting as direct CFTR chaperones, but in addition, Csp is positioned later in the CFTR biogenesis cascade where it regulates the production of mature CFTR by limiting its exit from the endoplasmic reticulum.
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PMID:Cysteine string protein monitors late steps in cystic fibrosis transmembrane conductance regulator biogenesis. 1646 39

All the alpha subunits of the Na+,K+ -ATPases and H+,K+ -ATPases have a protein kinase A (PKA) consensus sequence near or in the ninth transmembrane domain. The role of this domain in influencing alpha subunit synthesis/degradation, plasma membrane localization, and 86Rb+ uptake has not been established for the alpha subunit of the colonic H+,K+ -ATPase. This study examined the effect of mutating S955 (within the PKA consensus site of the alpha subunit of the colonic H+,K+ -ATPase [HKalpha2]) to alanine (S955/A) or aspartic acid (S955/D) on alpha subunit expression and function. The results demonstrate that a negatively charged amino acid at position 955 of HKalpha2 promotes higher expression levels of both whole-cell and plasma membrane-localized HKalpha2. Moreover, inhibition of PKA reduced expression of wild-type HKalpha2 and associated 86Rb+ uptake. Last, the activity of the HKalpha2 S955/A was rescued by treatment with 4-phenylbutyric acid, a compound that was shown previously to restore function to the cystic fibrosis transmembrane conductance regulator.
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PMID:Phosphorylation of S955 at the protein kinase A consensus promotes maturation of the alpha subunit of the colonic H+,K+ -ATPase. 1673 16

The photoreceptors lie between the inner retina and the retinal pigment epithelium (RPE). The release of glutamate by the phototoreceptors can signal changes in light levels to inner retinal neurons, but the role of glutamate in communicating with the RPE is unknown. Since RPE cells are known to release ATP, we asked whether glutamate could trigger ATP release from RPE cells and whether this altered cell signalling. Stimulation of the apical face of fresh bovine RPE eyecups with 100 mum NMDA increased ATP levels more than threefold, indicating that both receptors for NMDA and release of ATP occurred across the apical membrane of fresh RPE cells. NMDA increased ATP levels bathing cultured human ARPE-19 cells more than twofold, with NMDA receptor inhibitors MK-801 and d-AP5 preventing this release. Blocking the glycine site of the NMDA receptor with 5,7-dichlorokynurenic acid prevented ATP release from ARPE-19 cells. Release was also blocked by channel blocker NPPB and Ca(2+) chelator BAPTA, but not by cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide or vesicular release inhibitor brefeldin A. Glutamate produced a dose-dependent release of ATP from ARPE-19 cells that was substantially inhibited by MK-801. NMDA triggered a rise in cell Ca(2+) that was blocked by MK-801, by the ATPase apyrase, by the P2Y(1) receptor antagonist MRS2179 and by depletion of intracellular Ca(2+) stores with thapsigargin. These results suggest that glutamate stimulates NMDA receptors on the apical membrane of RPE cells to release ATP. This secondary release can amplify the glutaminergic signal by increasing Ca(2+) inside RPE cells, and might activate Ca(2+)-dependent conductances. The interplay between glutaminergic and purinergic systems may thus be important for light-dependent interactions between photoreceptors and the RPE.
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PMID:Glutamate acts at NMDA receptors on fresh bovine and on cultured human retinal pigment epithelial cells to trigger release of ATP. 1680 61


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