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 gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel thought to be critical for salt and water transport by epithelial cells. Plausible models exist to describe a role for ATP hydrolysis in CFTR channel activity; however, biochemical evidence that CFTR possesses intrinsic ATPase activity is lacking. In this study, we report the first measurements of the rate of ATP hydrolysis by purified, reconstituted CFTR. The mutation CFTRG551D resides within a motif conserved in many nucleotidases and is known to cause severe human disease. Following reconstitution the mutant protein exhibited both defective ATP hydrolysis and channel gating, providing direct evidence that CFTR utilizes ATP to gate its channel activity.
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PMID:ATPase activity of the cystic fibrosis transmembrane conductance regulator. 891 Apr 73

We have described a preparation of Necturus maculosus gallbladder (NGB) epithelium yielding isolated cells that retain structural and functional polarity ("figure-eight" cells). These cells have a normal membrane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled with hydrophobic fluorescent dyes; freeze-fracture electron microscopy reveals two distinct membrane domains separated by tight junctions; ZO-1, Na+/H+ exchanger (NHE3), and Na(+)-K(+)-ATPase are present in the junctional, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membrane domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 1996]. Here, we show that NGB epithelial cells express a protein cross-reactive with an antibody against human cystic fibrosis transmembrane conductance regulator (CFTR). In figure-eight cells, immunoreactivity was restricted to the apical membrane domain. Using intracellular microelectrodes and a novel method of regional superfusion, we found that control cells have high K+ conductances in both membranes and a small basolateral Cl- conductance, similar to findings in the epithelium. Activation of adenylate cyclase with forskolin elicited a large apical membrane Cl- conductance and membrane depolarization. Whole cell patch-clamp studies yielded a forskolin-activated linear Cl- current, with high Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells maintain the conductive membrane properties present in the epithelium, including polarized expression of adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductance is underlied by a CFTR homologue.
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PMID:Polarized expression of cAMP-activated chloride channels in isolated epithelial cells. 894 41

Immunofluorescence labelling and confocal microscopy were employed to examine the polarized distribution of several membrane transport proteins believed to be essential for salivary secretion in the rat submandibular gland. The Na+/K+-ATPase, Na+/H+ exchanger isoform 1 (NHE1), and the secretory Na+/K+/2Cl- cotransporter isoform were all found in the basolateral membranes of acinar and intralobular duct cells. Anion exchanger isoform 2 (AE2) was found only in the basolateral membranes of acinar cells, while AE1 was absent from glandular epithelial cells. Aquaporin 5 was detected in the apical membranes of acinar cells, while the cystic fibrosis transmembrane conductance regulator was found only in apical membranes of intralobular duct cells. NHEs 2 and 3 were found in the apical membranes of both acinar and intralobular duct cells. Our results are generally consistent with the expected distribution of most transporters based on previous physiological and pharmacological experiments. However, the apical localization of NHEs 2 and 3, and the presence of the secretory isoform of the Na+/K+/2Cl- cotransporter in intralobular duct cells were not predicted.
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PMID:Polarized distribution of key membrane transport proteins in the rat submandibular gland. 906 41

1. An improved novel plasmid backbone, pTrial10, has been developed. We have used this vector to deliver the cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR) to cells, both in vitro and in vivo, complexed with cationic liposomes. 2. Human 293 kidney epithelial cells (HEK 293) showed expression of an immunoprecipitable 165 kDa protein corresponding to CFTR when transfected in vitro with pTrial10-CFTR2, but not when the vector pTrial10 was used. 3. HEK 293 cells transfected with pTrial10-CFTR2, but not pTrial10, demonstrated a cAMP-dependent anion conductance, measured by fluorescence microscopy using a halide-sensitive probe, SPQ. 4. The CFTR-dependent, cAMP-sensitive chloride secretory response in murine tracheal epithelium could be measured if the calcium-dependent chloride secretory process was first maximally stimulated with a mixture of the Ca(2+)-ATPase inhibitor, TBHQ, and the calcium ionophore, A23187. With these conditions wild-type and CF-null (transgenic animals in which the cystic fibrosis (CF) gene has been disrupted so that no CFTR is produced) murine tracheas could be distinguished. The difference between the current elicited by forskolin in wild-type and CF tracheas was highly significantly different (P < 0.001), giving a CFTR-dependent current of 11.2 microA cm-2. 5. Transfection of the airways with pTrial10-CFTR2, but not pTrial10, significantly (P < 0.01) increased the CFTR-dependent chloride secretory current in CF tracheas. The degree of correction was greater when intra-tracheal installation rather than nasal insufflation was used to deliver the plasmids.
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PMID:Chloride secretion in the trachea of null cystic fibrosis mice: the effects of transfection with pTrial10-CFTR2. 913 Jan 64

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

The cystic fibrosis transmembrane conductance regulator (CFTR) forms a tightly regulated channel that mediates the passive diffusion of Cl- ions. Here we show, using macroscopic current recording from excised membrane patches, that CFTR also shows significant, but highly asymmetrical, permeability to a broad range of large organic anions. Thus, all large organic anions tested were permeant when present in the intracellular solution under biionic conditions (PX/PCl = 0.048-0.25), whereas most were not measurably permeant when present in the extracellular solution. This asymmetry was not observed for smaller anions. ATPase inhibitors that "lock" CFTR channels in the open state (pyrophosphate, 5'-adenylylimidodiphosphate) disrupted the asymmetry of large anion permeation by allowing their influx from the extracellular solution, which suggests that ATP hydrolysis is required to maintain asymmetric permeability. The ability of CFTR to allow efflux of large organic anions represents a novel function of CFTR. Loss of this function may contribute to the pleiotropic symptoms seen in cystic fibrosis.
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PMID:Adenosine triphosphate-dependent asymmetry of anion permeation in the cystic fibrosis transmembrane conductance regulator chloride channel. 952 41

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the traffic ATPase family that includes multiple proteins characterized by (1) ATP binding, (2) conserved transmembrane (TM) motifs and nucleotide binding domains (NBDs), and (3) molecular transport of small molecules across the cell membrane. While CFTR NBD-1 mediates ATP binding and hydrolysis, the membrane topology and function of this domain in living eukaryotic cells remains uncertain. In these studies, we have expressed wild-type CFTR NBD-1 (amino acids 433-586) or NBD-1 containing the DeltaF508 mutation transiently in COS-7 cells and established that the domain is situated across the plasma membrane by four independent assays; namely, extracellular chymotrypsin digestion, surface protein biotinylation, confocal immunofluorescent microscopy, and functional measurements of cell membrane anion permeability. Functional studies indicate that basal halide permeability is enhanced above control conditions following wild-type or DeltaF508 NBD-1 expression in three different epithelial cell lines. Furthermore, when clinically relevant CFTR proteins truncated within NBD-1 (R553X or G542X) are expressed, surface localization and enhanced halide permeability are again established. Together, these findings suggest that isolated CFTR NBD-1 (with or without the DeltaF508 mutation) is capable of targeting the epithelial cell membrane and enhancing cellular halide permeability. Furthermore, CFTR truncated at position 553 or 542 and possessing the majority of NBD-1 demonstrates surface localization and also confers increased halide permeability. These findings indicate that targeting to the plasma membrane and assumption of a transmembrane configuration are innate properties of the CFTR NBD-1. The results also support the notion that components of the halide-selective pore of CFTR reside within NBD-1.
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PMID:Cystic fibrosis transmembrane conductance regulator (CFTR) nucleotide-binding domain 1 (NBD-1) and CFTR truncated within NBD-1 target to the epithelial plasma membrane and increase anion permeability. 979 Jun 86

Natriuretic peptides (NP) act as ligands on the guanylyl cyclase family of receptors. The NP binding site on these receptors is extracellular and the guanylyl cyclase and protein kinase domains are intracellular. The guanylyl cyclase receptor catalyzes the synthesis of the second messenger molecule, cGMP, which activates protein kinase. This in turn is involved in the phosphorylation of various ion transport proteins. Ion transport proteins, which are modulated by NP and are thought to underlie the natriuretic and diuretic actions of NP, include: (a) calcium-activated K+ channels; (b) ATP-sensitive K+ channels; (c) inwardly-rectifying K+ channels; (d) outwardly-rectifying K+ channels; (e) L-type Ca2+ channels; (f) Cl- channels including cystic fibrosis transmembrane conductance regulator Cl- channels; (g) Na+- K+ 2Cl- co-transporter; (h) Na+- K+ ATPase; (i) Na+ channels; (j) stretch-activated channels; and (k) water channels. It appears that NP modulate the kinetics, rather than the conductance, of ion channels. Some of these channels, like the Ca2+, ATP-sensitive K+ and stretch-activated channels, are also involved in NP secretion. In addition, the structural properties of the NP, e.g., ovCNP-22 and ovCNP-39, appear to confer on them the ability to form ion channels. These CNP-formed ion channels can modify the trans-membrane signal transduction and second messenger systems underlying NP-induced pathological effects.
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PMID:Role of natriuretic peptides in ion transport mechanisms. 991 94

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


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