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 colonic epithelium has both absorptive and secretory functions. The transport is characterized by a net absorption of NaCl, short-chain fatty acids (SCFA), and water, allowing extrusion of a feces with very little water and salt content. In addition, the epithelium does secret mucus, bicarbonate, and KCl. Polarized distribution of transport proteins in both luminal and basolateral membranes enables efficient salt transport in both directions, probably even within an individual cell. Meanwhile, most of the participating transport proteins have been identified, and their function has been studied in detail. Absorption of NaCl is a rather steady process that is controlled by steroid hormones regulating the expression of epithelial Na(+) channels (ENaC), the Na(+)-K(+)-ATPase, and additional modulating factors such as the serum- and glucocorticoid-regulated kinase SGK. Acute regulation of absorption may occur by a Na(+) feedback mechanism and the cystic fibrosis transmembrane conductance regulator (CFTR). Cl(-) secretion in the adult colon relies on luminal CFTR, which is a cAMP-regulated Cl(-) channel and a regulator of other transport proteins. As a consequence, mutations in CFTR result in both impaired Cl(-) secretion and enhanced Na(+) absorption in the colon of cystic fibrosis (CF) patients. Ca(2+)- and cAMP-activated basolateral K(+) channels support both secretion and absorption of electrolytes and work in concert with additional regulatory proteins, which determine their functional and pharmacological profile. Knowledge of the mechanisms of electrolyte transport in the colon enables the development of new strategies for the treatment of CF and secretory diarrhea. It will also lead to a better understanding of the pathophysiological events during inflammatory bowel disease and development of colonic carcinoma.
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PMID:Electrolyte transport in the mammalian colon: mechanisms and implications for disease. 1177 14

The cystic fibrosis transmembrane conductance regulator (CFTR), which is aberrant in patients with cystic fibrosis, normally functions both as a chloride channel and as a pleiotropic regulator of other ion transporters. Here we show, by ratiometric imaging with luminally exposed pH-sensitive green fluorescent protein, that CFTR affects the pH of cellubrevin-labeled endosomal organelles resulting in hyperacidification of these compartments in cystic fibrosis lung epithelial cells. The excessive acidification of intracellular organelles was corrected with low concentrations of weak base. Studies with proton ATPase and sodium channel inhibitors showed that the increased acidification was dependent on proton pump activity and sodium transport. These observations implicate sodium efflux in the pH homeostasis of a subset of endocytic organelles and indicate that a dysfunctional CFTR in cystic fibrosis leads to organellar hyperacidification in lung epithelial cells because of a loss of CFTR inhibitory effects on sodium transport. Furthermore, recycling of transferrin receptor was altered in CFTR mutant cells, suggesting a previously unrecognized cellular defect in cystic fibrosis, which may have functional consequences for the receptors on the plasma membrane or within endosomal compartments.
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PMID:Hyperacidification of cellubrevin endocytic compartments and defective endosomal recycling in cystic fibrosis respiratory epithelial cells. 1180 65

This paper reviews experiments from this lab that have tested the hypothesis that pH of the Golgi (pH(G)) of cystic fibrosis (CF) airway epithelial cells is alkaline compared to normal, that this altered pH affects sialyltransferase and other Golgi enzymes controlling biochemical composition of the plasma membrane and that altered surface biochemistry increases bacterial binding. We generated a plasmid encoding a modified green fluorescence protein-sialyltransferase (GFP-ST) chimera protein that was pH-sensitive and localized to the Golgi when transfected into HeLa cells and also CF and normal or cystic fibrosis transmembrane conductance regulator- (CFTR)-corrected airway epithelial cells. Digital imaging microscopy of these Golgi-localized probes showed that there was no correlation between pH(G) (6.4-7.0) and the presence of CFTR, whether cells were in HCO(3)(-)/CO(2)-containing or in HCO(3)(-)/CO(2)-free solutions. Activation of CFTR by raising cell [cAMP] had no effect on pH(G). Thus, CFTR seemed not to be involved in controlling pH(G). Experiments on HeLa cells using an avidin-sialyltransferase chimera in combination with a pH-sensitive fluorescent biotin indicated that even in cells that do not express CFTR, Cl(-) and K(+) conductances of the Golgi and other organelle membranes were large and that pH(G) was controlled solely by the H(+) v-ATPase countered by a H(+) leak. A mathematical model was applied to these and other published data to calculate passive H(+) permeability (P(H+)) of the Golgi, endoplasmic reticulum, trans-Golgi network, recycling endosomes and secrety granules from a variety of cells. An organelle's acidity was inversely correlated to its calculated P(H+). We conclude that the CFTR plays a minor role in organelle pH regulation because other (Cl(-) and K(+)) channels are present in sufficient numbers to shunt voltages generated during H(+) pumping. Acidity of the Golgi (and perhaps other organelles) appears to be determined by the activity of H(+) pumps countered by H(+) leaks.
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PMID:Cystic fibrosis transmembrane conductance regulator and H+ permeability in regulation of Golgi pH. 1187 64

To test for the presence of HCO(3)(-) transport across airway epithelia, we measured short-circuit current in primary cultures of canine and human airway epithelia bathed in a Cl(-)-free, HCO(3)(-)/CO(2)-buffered solution. cAMP agonists stimulated a secretory current that was likely carried by HCO(3)(-) because it was absent in HCO(3)(-)-free solutions. In addition, the cAMP-stimulated current was inhibited by the carbonic anhydrase inhibitor, acetazolamide, and by the apical addition of a blocker of cystic fibrosis transmembrane conductance regulator (CFTR), diphenylamine-2-carboxylate. The current was dependent on Na(+) because it was inhibited by removing Na(+) from the submucosal solution and by inhibition of the Na(+)-K(+)-ATPase with ouabain. The cAMP-stimulated current was absent in cystic fibrosis (CF) airway epithelia. These data suggest that cAMP agonists can stimulate HCO(3)(-) secretion across airway epithelia and that CFTR may provide a conductive pathway for HCO(3)(-) movement across the apical membrane.
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PMID:cAMP stimulation of HCO3- secretion across airway epithelia. 1187 74

The discovery of mechanisms that regulate salt and water transport by the alveolar and distal airway epithelium of the lung has generated new insights into the regulation of lung fluid balance under both normal and pathological conditions. There is convincing evidence that active sodium and chloride transporters are expressed in the distal lung epithelium and are responsible for the ability of the lung to remove alveolar fluid at the time of birth as well as in the mature lung when pathological conditions lead to the development of pulmonary edema. Currently, the best described molecular transporters are the epithelial sodium channel, the cystic fibrosis transmembrane conductance regulator, Na+-K+-ATPase, and several aquaporin water channels. Both catecholamine-dependent and -independent mechanisms can upregulate isosmolar fluid transport across the distal lung epithelium. Experimental and clinical studies have made it possible to examine the role of these transporters in the resolution of pulmonary edema.
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PMID:Lung epithelial fluid transport and the resolution of pulmonary edema. 1208 29

Resolution of pulmonary edema involved active transepithelial sodium transport. Although several of the cellular and molecular mechanisms involved are relatively well understood, it is only recently that the regulation of these mechanisms in injured lung are being evaluated. Interestingly, in mild-to-moderate lung injury, alveolar edema fluid clearance is often preserved. This preserved or enhanced alveolar fluid clearance is mediated by catecholamine-dependent or -independent mechanisms. This stimulation of alveolar liquid clearance is related to activation or increased expression of sodium transport molecules such as the epithelial sodium channel or the Na(+)-K(+)-ATPase pump and may also involve the cystic fibrosis transmembrane conductance regulator. When severe lung injury occurs, the decrease in alveolar liquid clearance may be related to changes in alveolar permeability or to changes in activity or expression of sodium or chloride transport molecules. Multiple pharmacological tools such as beta-adrenergic agonists, vasoactive drugs, or gene therapy may prove effective in stimulating the resolution of alveolar edema in the injured lung.
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PMID:Lung edema clearance: 20 years of progress: invited review: alveolar edema fluid clearance in the injured lung. 1243 40

Na,K-ATPase, an alpha, beta heterodimer, is found in the plasma membrane of all animal cells. The alpha chain is believed to have 10 transmembrane regions and a large cytoplasmic domain between the 4th and 5th transmembrane regions (H4-H5). In our previous report, the large (3rd) cytoplasmic domains of the alpha1 and alpha2 isoform were found to interact with cofilin, an actin-modulating protein, by the yeast two-hybrid system. Here we show that cofilin interacts only with the 3rd cytoplasmic domain of the alpha2 subunit but not with the 2nd, 4th, and 5th cytoplasmic domains or the cytoplasmic region of the beta subunit of Na,K-ATPase. We also demonstrate that cofilin interacts with the large cytoplasmic domains of the alpha1, alpha2 and alpha3 isoforms of Na,K-ATPase, but not with those of glucose transporter 1, glucose transporter 4, cystic fibrosis transmembrane conductance regulator and plasma membrane Ca-ATPase. We introduced 10 mutations into the 3rd cytoplasmic domain of Na,K-ATPase to identify the binding sites with cofilin. Eight of these mutants were single amino acid substitutions (R417Q, K470Q, K654G, D672A, K691A, R700G, R700A and D710G) and two were double mutant (K654GR700G and K719AK720A). Analysis of the activity of the reporter gene of these mutants shows that residues D672 and R700 of the 3rd cytoplasmic domain of Na,K-ATPase are involved in the interaction with cofilin.
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PMID:Identification of the cofilin-binding sites in the large cytoplasmic domain of Na,K-ATPase. 1250 82

Cystic fibrosis (CF) is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator), a regulated anion channel and member of the ATP-binding-cassette transporter (ABC transporter) superfamily. Of CFTR's five domains, the first nucleotide-binding fold (NBF1) has been of greatest interest both because it is the major 'hotspot' for mutations that cause CF, and because it is connected to a unique regulatory domain (R). However, attempts have failed to obtain a catalytically active NBF1+R protein in the absence of a fusion partner. Here, we report that such a protein can be obtained following its overexpression in bacteria. The pure NBF1+R protein exhibits significant ATPase activity [catalytic-centre activity (turnover number) 6.7 min(-1)] and an apparent affinity for ATP ( K (m), 8.7 microM) higher than reported previously for CFTR or segments thereof. As predicted, the ATPase activity is inhibited by mutations in the Walker A motif. It is also inhibited by vanadate, a transition-state analogue. Surprisingly, however, the best divalent metal activator is Co(2+), followed by Mn(2+) and Mg(2+). In contrast, Ca(2+) is ineffective and Cd(2+) is a potent inhibitor. These novel studies, while demonstrating clearly that CFTR's NBF1+R segment can act independently as an active, vanadate-sensitive ATPase, also identify its unique cation activators and a new inhibitor, thus providing insight into the nature of its active site.
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PMID:Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-binding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2+-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanadate. 1252 35

Previous studies have shown that alpha2 adrenoceptor (alpha2AR) agonists inhibit electrolyte secretion in colonic epithelia, but little is known about the molecular mechanisms involved in this process. In this study we examined the effect of alpha2AR activation on transepithelial anion secretion across isolated murine colonic epithelium. We found that alpha2AR agonists, UK 14,304, clonidine and medetomidine were potent inhibitors of anion secretion, especially in the proximal colon. Short circuit current measurements (Isc) in colonic epithelia from normal and cystic fibrosis (CF) mice showed that alpha2AR agonists inhibited basal cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion but had no effect on CFTR activation by cAMP-dependent phosphorylation. Apical administration of an ionophore, nystatin (90 microg ml-1), was used to investigate the effect of UK 14,304 on basolateral K+ transport. The Na+-K+-ATPase current, measured as ouabain-sensitive current in the absence of ion gradients, was unaltered by pretreatment of the tissue with UK 14,304 (1 microM). In the presence of a basolaterally directed K+ gradient, UK 14,304 significantly reduced nystatin-activated Isc indicating that activation of alpha2ARs inhibits basolateral K+ channels. Studies with selective K+ channel inhibitors and openers showed that alpha2AR agonists inhibited KATP channels that were tonically active in mouse colonic epithelia. RT-PCR and pharmacological studies suggested that these channels could be similar to vascular smooth muscle KATP channels comprising Kir6.1/SUR2B or Kir6.2/SUR2B subunits. Inhibition of anion secretion by alpha2AR agonists required activation of pertussis toxin-sensitive Gi/o proteins, but did not involve classical second messengers, such as cAMP or Ca2+. In summary, alpha2ARs inhibit anion secretion in colonic epithelia by acting on basolateral KATP channels, through a process that does not involve classical second messengers.
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PMID:Regulation of Cl- secretion by alpha2-adrenergic receptors in mouse colonic epithelium. 1259 92

Gating of the CFTR Cl- channel is associated with ATP hydrolysis at the nucleotide-binding domains (NBD1, NBD2) and requires PKA (protein kinase A) phosphorylation of the R domain. The manner in which the NBD1, NBD2 and R domains of CFTR (cystic fibrosis transmembrane conductance regulator) interact to achieve a properly regulated ion channel is largely unknown. In this study we used bacterially expressed recombinant proteins to examine interactions between these soluble domains of CFTR in vitro. PKA phosphorylated a fusion protein containing NBD1 and R (NBD1-R-GST) on CFTR residues Ser-660, Ser-700, Ser-712, Ser-737, Ser-768, Ser-795 and Ser-813. Phosphorylation of these serine residues regulated ATP hydrolysis by NBD1-R-GST by increasing the apparent K(m) for ATP (from 70 to 250 microM) and the Hill coefficient (from 1 to 1.7) without changing the V(max). When fusion proteins were photolabelled with 8-azido-[alpha-32P]ATP, PKA phosphorylation increased the apparent k(d) for nucleotide binding and it caused binding to become co-operative. PKA phosphorylation also resulted in dimerization of NBD1-R-GST but not of R-GST, a related fusion protein lacking the NBD1 domain. Finally, an MBP (maltose-binding protein) fusion protein containing the NBD2 domain (NBD2-MBP) associated with and regulated the ATPase activity of PKA-phosphorylated NBD1-R-GST. Thus when the R domain in NBD1-R-GST is phosphorylated by PKA, ATP binding and hydrolysis becomes co-operative and NBD dimerization occurs. These findings suggest that during the activation of native CFTR, phosphorylation of the R domain by PKA can control the ability of the NBD1 domain to hydrolyse ATP and to interact with other NBD domains.
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PMID:Protein kinase A regulates ATP hydrolysis and dimerization by a CFTR (cystic fibrosis transmembrane conductance regulator) domain. 1460 47


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