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)

The role of epithelial ion transport in the activation of water-responsive laryngeal afferent was investigated in anesthetized, spontaneously breathing cats. Single-fiber recordings from the peripheral cut-end of the superior laryngeal nerve were carried out to identify water-responsive laryngeal afferent. Substitution of chloride ions (Cl-) of the Krebs solution with gluconate activated the water-responsive endings when the gluconate concentration was > or = 50 mM. Amiloride (10(-4), 10(-3) and 10(-2) M), an inhibitor of epithelial sodium channels, reduced the water-responsiveness of these afferents, whereas EIPA (5 x 10(-5) M), an amiloride analogue which inhibits Na+/H+ exchange, had no effect. Both ouabain (10(-4) M), an inhibitor of Na+/K+ ATPase, and bumetanide (10(-4) M), an inhibitor of Na(+)-K(+)-2Cl- cotransport, reduced the water response, but no significant reduction in the response was observed with DIDS and DPC, two chloride channel inhibitors. These findings suggest that the epithelium modulates the water-responsiveness of laryngeal afferent but is not the primary determinant of the response.
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PMID:Activation of water-responsive laryngeal afferents: role of epithelial ion transport. 893 Nov 75

The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca2+]i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca2+]i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 microM) increased [Ca2+]i from 66 +/- 9 to 137 +/- 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (Isc) to drop from 26.4 to 10.6 microA cm-2 (n = 19, P<0.001) concomitant with a depolarization of the cells from -79 +/- 1 to -31 +/- 2 mV (n = 18, P<0.001). Apical sodium permeability (PaNa) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. PaNa decreased from 8.01.10(-7 )to 3.74.10(-7) cm s-1 (n = 7, P = 0.04) following an increase in [Ca2+]i. A decrease in apical sodium permeability per se would tend to decrease Isc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca2+]i. This was confirmed in wash-out experiments with 36Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 +/- 5.01 to 62.63 +/- 13.3 nmol.min-1 cm-2 (n = 5, P = 0.04). We conclude that a small increase in [Ca2+]i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption.
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PMID:An increase in [Ca2+]i activates basolateral chloride channels and inhibits apical sodium channels in frog skin epithelium. 901 17

This report describes a novel, single-step strategy for the purification of the cystic fibrosis transmembrane conductance regulator from Sf9 cells, which will facilitate studies of the structure-function relationships of this clinically important molecule. The new method combines the use of the novel detergent sodium pentadecafluoro-octanoate with metal-affinity chromatography to produce a high yield of purified protein which can be functionally reconstituted as a chloride channel and an ATPase.
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PMID:A novel procedure for the efficient purification of the cystic fibrosis transmembrane conductance regulator (CFTR). 935 28

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel situated on the apical membrane of epithelial cells. Our recent studies of purified, reconstituted CFTR revealed that it also functions as an ATPase and that there may be coupling between ATP hydrolysis and channel gating. Both the ATP turnover rate and channel gating are slow, in the range of 0.2 to 1 s(-1), and both activities are suppressed in a disease-causing mutation situated in a putative nucleotide binding motif. Our future studies using purified protein will be directed toward understanding the structural basis and mechanism for coupling between hydrolysis and channel function.
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PMID:Coupling of ATP hydrolysis with channel gating by purified, reconstituted CFTR. 951 31

A defect in the yeast GEF1 gene, a CLC chloride channel homolog leads to an iron requirement and cation sensitivity. The iron requirement is due to a failure to load Cu2+ onto a component of the iron uptake system, Fet3. This process, which requires both Gef1 and the Menkes disease Cu2+-ATPase yeast homolog Ccc2, occurs in late- or post-Golgi vesicles, where Gef1 and Ccc2 are localized. The defects of gef1 mutants can be suppressed by the introduction of Torpedo marmorata CLC-0 or Arabidopsis thaliana CLC-c and -d chloride channel genes. The functions of Gef1 in cation homeostasis provide clues to the understanding of diseases caused by chloride channel mutations in humans and cation toxicity in plants.
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PMID:The yeast CLC chloride channel functions in cation homeostasis. 952 Apr 90

Loss-of-function mutations of the ClC-5 chloride channel lead to Dent's disease, a syndrome characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones. We show that ClC-5 is expressed in renal proximal tubule cells, which normally endocytose proteins passing the glomerular filter. Expression is highest below the brush border in a region densely packed with endocytotic vesicles, where ClC-5 colocalizes with the H+-ATPase and with internalized proteins early after uptake. In intercalated cells of the collecting duct it again localizes to apical intracellular vesicles and colocalizes with the proton pump in alpha-intercalated cells. In transfected cells, ClC-5 colocalizes with endocytosed alpha2-macroglobulin. Cotransfection with a GTPase-deficient rab5 mutant leads to enlarged early endosomes that stain for ClC-5. We suggest that ClC-5 may be essential for proximal tubular endocytosis by providing an electrical shunt necessary for the efficient acidification of vesicles in the endocytotic pathway, explaining the proteinuria observed in Dent's disease.
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PMID:ClC-5, the chloride channel mutated in Dent's disease, colocalizes with the proton pump in endocytotically active kidney cells. 965 42

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATPase and as a chloride channel. It has been hypothesized, on the basis of electrophysiological findings, that the catalytic activity of CFTR is tightly coupled to the opening and closing of the channel gate. In the present study, to determine the structural basis for the ATPase activity of CFTR, we assessed the effect of mutations within the "Walker A" consensus motifs on ATP hydrolysis by the purified, intact protein. Mutation of the lysine residue in the "Walker A" motif of either the first nucleotide binding fold (CFTRK464A) or the second nucleotide binding fold (CFTRK1250A) inhibited the ATPase activity of the purified intact CFTR protein significantly, by greater than 50%. This finding suggests that the two nucleotide binding folds of CFTR are functioning cooperatively in catalysis. However, the rate of channel gating was only significantly inhibited in one of these purified mutants, CFTRK1250A, suggesting that ATPase activity may not be tightly coupled to channel gating as previously hypothesized.
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PMID:Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator). 993 Oct 11

The function of the human cystic fibrosis transmembrane conductance regulator (CFTR) protein as a chloride channel or transport regulator involves cellular ATP binding and cleavage. Here we describe that human CFTR expressed in insect (Sf9) cell membranes shows specific, Mg2+-dependent nucleotide occlusion, detected by covalent labeling with 8-azido-[alpha-32P]ATP. Nucleotide occlusion in CFTR requires incubation at 37 degrees C, and the occluded nucleotide can not be removed by repeated washings of the membranes with cold MgATP-containing medium. By using limited tryptic digestion of the labeled CFTR protein we found that the adenine nucleotide occlusion preferentially occurred in the N-terminal nucleotide binding domain (NBD). Addition of the ATPase inhibitor vanadate, which stabilizes an open state of the CFTR chloride channel, produced an increased nucleotide occlusion and resulted in the labeling of both the N-terminal and C-terminal NBDs. Protein modification with N-ethylmaleimide prevented both vanadate-dependent and -independent nucleotide occlusion in CFTR. The pattern of nucleotide occlusion indicates significant differences in the ATP hydrolyzing activities of the two NBDs, which may explain their different roles in the CFTR channel regulation.
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PMID:Nucleotide occlusion in the human cystic fibrosis transmembrane conductance regulator. Different patterns in the two nucleotide binding domains. 1021 85

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic adenosine monophosphate dependent, low-conductance chloride channel found on the apical plasma membrane of secretory epithelia. Surprisingly, since cystic fibrosis patients have no kidney phenotype, CFTR is highly expressed in the kidney, present from 12 weeks of gestation in the human metanephric kidney. As well as the mature, full-length, 165-kD wild-type protein (WT-CFTR) associated with renal tubule plasma membranes, intracellular, partially glycosylated forms are also seen in normal kidneys. In addition, a kidney-specific splice variant of CFTR translates a cytoplasmic truncated protein (TNR-CFTR), apparently associated with a specific small endosomal population, and is predominantly expressed in the renal medulla. WT-CFTR and TNR-CFTR show different patterns of developmental regulation, WT-CFTR being the major form expressed early in metanephric development when it is localized at the apical plasma membrane of developing collecting tubules. By contrast, TNR-CFTR expression increases with gestational age, reaching adult levels at 23 weeks. Evidence suggests that WT-CFTR plays a role in chloride secretion into the apical lumen of normal distal tubules. In autosomal dominant polycystic kidney disease, normally targeted CFTR at the apical plasma membrane in association with mislocalized Na-K-ATPase may result in abnormal fluid secretion into cysts. Similar colocalization of WT-CFTR and Na-K-ATPase at the apical plasma membranes is found in collecting tubules during development when it is speculated to play a role in the initiation of opening of the tubule lumen.
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PMID:Cystic fibrosis transmembrane conductance regulator in the kidney: clues to its role? 1045 15

Residues 417-830 of the cystic fibrosis transmembrane conductance regulator (CFTR) were expressed as a glutathione-S-transferase fusion protein. This fusion protein, NBD1/R/GST, contains the regulatory and first nucleotide binding domains of CFTR. NBD1/R/GST hydrolyzed ATP with a K(M) (60 microM) and V(max) (330 nmol/min/mg) that differed from those reported for CFTR and for a peptide containing CFTR residues 433-589. The ATPase inhibitor profile of NBD1/R/GST indicates that CFTR resembles P-glycoprotein with respect to the NBD1 ATPase catalytic mechanism. ATP hydrolysis by NBD1/R/GST was unaffected by genistein, glybenclamide, and other agents known to affect CFTR's chloride channel function, suggesting that these agents do not act by directly influencing the ATPase function of NBD1. The disease-causing mutation, G551D, reduced ATP hydrolysis by NBD1/R/GST by increasing the K(M) for ATP fourfold. This suggests that when G551D occurs in patients with cystic fibrosis, it affects CFTR function by reducing the affinity of NBD1 for ATP.
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PMID:ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation. 1079 28


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